Substituted CC-1065 analogs and their conjugates

ABSTRACT

This invention relates to novel agents that are analogs of the DNA-alkylating agent CC-1065 and to their conjugates. Furthermore this invention concerns intermediates for the preparation of said agents and their conjugates. The conjugates are designed to release their (multiple) payload after one or more activation steps and/or at a rate and time span controlled by the conjugate in order to selectively deliver and/or controllably release one or more of said DNA alkylating agents. The agents, conjugates, and intermediates can be used to treat an illness that is characterized by undesired (cell) proliferation. As an example, the agents and the conjugates of this invention may be used to treat a tumor.

This application is a divisional of U.S. application Ser. No.12/671,609, filed Oct. 26, 2010, which was the national stage of PCTapplication serial number PCT/NL2007/050384, filed Aug. 1, 2007; theentire contents of these prior applications being incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to novel agents that are analogs of theDNA-alkylating agent CC-1065 and to their conjugates. Furthermore thisinvention concerns intermediates for the preparation of said agents andtheir conjugates. The conjugates are designed to release their(multiple) payload after one or more activation steps and/or at a rateand time span controlled by the conjugate in order to selectivelydeliver and/or controllably release one or more of said DNA alkylatingagents. The agents, conjugates, and intermediates can be used to treatan illness that is characterized by undesired (cell) proliferation. Asan example, the agents and the conjugates of this invention may be usedto treat a tumor.

BACKGROUND OF THE INVENTION

The duocarmycins, first isolated from a culture broth of Streptomycesspecies, are members of a family of antitumor antibiotics that alsoincludes CC-1065. These extremely potent agents allegedly derive theirbiological activity from an ability to sequence-selectively alkylate DNAat the N3 of adenine in the minor groove, which initiates a cascade ofevents that terminates in an apoptotic cell death mechanism.¹

Although CC-1065 has shown very potent cytotoxicity, it could not beused in the clinic because of serious delayed hepatotoxicity.² Thisobservation led to the development of synthetic analogs of CC-1065 (seefor CC-1065 derivatives for example Aristoff et al., J. Org. Chem. 1992,57, 6234; Boger et al., Bioorg. Med. Chem. Lett. 1996, 6, 2207; Boger etal., Chem. Rev. 1997, 97, 787; Milbank et al., J. Med. Chem. 1999, 42,649; Atwell et al., J. Med. Chem. 1999, 42, 3400; Wang et al., J. Med.Chem. 2000, 43, 1541; Boger et al., Bioorg. Med. Chem. Lett 2001, 11,2021; Parrish et al., Bioorg. Med. Chem. 2003, 11, 3815; Danicll et al.,Bioorg. Med. Chem. Lett. 2005, 15, 177; Tichenor et al., J. Am. Chem.Soc. 2006, 128, 15683; Purnell et al., Bioorg. Med. Chem. 2006, 16,5677; Bando and Sugiyama, Acc. Chem. Res. 2006, 39, 935; EP0154445; WO88/04659; WO 90/02746; WO 97/12862; WO 97/32850; WO 97/45411; WO98/52925; WO 99/19298; WO 01/83482; WO 02/067937; WO 02/067930; WO02/068412; WO 03/022806; WO 2004/101767; WO 2006/043839; and WO2007/051081), which generally showed to have similar cytotoxicity, butreduced hepatotoxicity. Still, however, these derivatives lacksufficient selectivity for tumor cells as the selectivity of theseagents—and cytotoxic agents in general—is for a considerable part basedon the difference in the rate of proliferation of tumor cells and normalcells, and therefore they also affect healthy cells that show arelatively high proliferation rate. This typically leads to severe sideeffects. Drug concentrations that would completely eradicate the tumorcannot be reached because of dose-limiting side effects such asgastrointestinal tract and bone marrow toxicity. In addition, tumors candevelop resistance against anticancer agents after prolonged treatment.In modern drug development, targeting of cytotoxic drugs to the tumorsite can be considered one of the primary goals.

One promising approach to obtain selectivity for tumor cells or tumortissue is to exploit the existence of tumor-associated antigens,receptors, and other receptive moieties, which can serve as a target.Such a target may be upregulated or to some degree be specificallypresent in tumor tissue or in closely associated tissue, such asneovascular tissue, with respect to other tissues in order to achieveefficient targeting. Many targets have been identified and validated andseveral methods to identify and validate targets have been developed.³By coupling a ligand, e.g. an antibody or antibody fragment, for such atumor-associated antigen, receptor, or other receptive moiety to atherapeutic agent, this agent can be selectively targeted to tumortissue.

Another promising approach to obtain selectivity for tumor cells ortumor tissue is to exploit the existence of tumor-associated enzymes. Anenzyme that is mainly localized at the tumor site can convert apharmacologically inactive prodrug, which consists of an enzymesubstrate directly or indirectly linked to the toxic drug, to thecorresponding drug in the vicinity of or inside the tumor.

Via this concept a high concentration of toxic anticancer agent can beselectively generated at the tumor site. All tumor cells may be killedif the dose is sufficiently high, which may decrease development ofdrug-resistant tumor cells.

Enzymes can also be transported to the vicinity of or inside targetcells or target tissue via for example antibody-directed enzyme prodrugtherapy (ADEPT)⁴, polymer-directed enzyme prodrug therapy (PDEPT) ormacromolecular-directed enzyme prodrug therapy (MDEPT)⁵, virus-directedenzyme prodrug therapy (VDEPT)⁶, or gene-directed enzyme prodrug therapy(GDEPT)⁷. With ADEPT, for example, a non-toxic prodrug is selectivelyconverted into a cytotoxic compound at the surface of target cells by anantibody-enzyme conjugate that has been pretargeted to the surface ofthose cells.

Yet another promising approach to obtain selectivity for tumor cells ortumor tissue is to exploit the enhanced permeability and retention(EPRB) effect. Through this EPR effect, macromolecules passivelyaccumulate in solid tumors as a consequence of the disorganizedpathology of angiogenic tumor vasculature with its discontinuousendothelium, leading to hyperpermeability to large macromolecules, andthe lack of effective tumor lymphatic drainage.⁸ By coupling atherapeutic agent directly or indirectly to a macromolecule, said agentcan be selectively targeted to tumor tissue.

Besides efficient targeting, other important criteria for the successfulapplication of targeted conjugates of cytotoxic agents in tumor therapyare that the one or more agents are released efficiently from theconjugate at the tumor site and that the conjugate is non-cytotoxic oronly very weakly cytotoxic, whereas the cytotoxic agent itself exhibitshighly potent cytotoxicity. Ideally, this leads to the generation ofcytotoxic molecules only at the tumor site, which results in a greatlyincreased therapeutic index with respect to the untargeted cytotoxicagent. Another important criterion for a successful targeted conjugateis that the conjugate must have suitable pharmacological properties,such as sufficient stability in the circulation, low aggregationtendency, and good water solubility.

Several conjugates of CC-1065 and derivatives have been described (seefor conjugates of CC-1065 derivatives for example Suzawa et al., Bioorg.Med. Chem. 2000, 8, 2175; Jeffrey et al., J. Med. Chem. 2005, 48, 1344;Wang et al., Bioorg. Med Chem. 2006, 14, 7854; Tietze et al., Chem. Eur.J. 2007, 13, 4396; WO 91/16324; WO 94/04535; WO 95/31971; U.S. Pat. No.5,475,092; U.S. Pat. No. 5,585,499; U.S. Pat. No. 5,646,298; WO97/07097; WO 97/44000; U.S. Pat. No. 5,739,350; WO 98/11101; WO98/25898; U.S. Pat. No. 5,843,937; U.S. Pat. No. 5,846,545; WO02/059122; WO 02/30894; WO 03/086318; WO 2005/103040; WO 2005/112919; WO2006/002895; WO 2006/110476; WO 2007/038658; and WO 2007/059404). Inthese conjugates, one or more of the favorable properties discussedabove are non-optimal. As an illustrative example, glycoside conjugatesof seco CC-1065 analogs have been described that can be activated at thelesion site via an ADEPT approach.⁹ The difference in cytotoxicitybetween the conjugates and the corresponding drugs, expressed as thecytotoxicity quotient, IC_(50, conjugate)/IC_(50, parent drug), washowever relatively low, and the seco CC-1065 analogs themselves did notshow extremely potent cytotoxicity. Improvements to the cytotoxicityquotient were made through the development of glycoside conjugates ofseco CC-1065 derivatives with a secondary leaving group.¹⁰ Althoughthese conjugates demonstrated a high cytotoxicity quotient, theirpharmacological properties were non-optimal. For instance, theygenerally showed poor water solubility, which is a consequence of theinherent lipophilic nature of the CC-1065 class of compounds.

Accordingly, there is still a clear need in the art for conjugates ofCC-1065 derivatives that show high cytotoxicity quotients, containCC-1065 derivatives that have potent cytotoxicity and favorablepharmacological properties, and release the CC-1065 derivativesefficiently.

SUMMARY OF THE INVENTION

The present invention fulfils the above-mentioned need with a compoundof formula (I) or (II):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinR¹ is a leaving group;R² is selected from H, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH,halogen, R^(b), SR^(b), S(O)R^(a), S(O)₂R^(a), S(O)OR^(a), S(O)₂OR^(a),OS(O)R^(a), OS(O)₂R^(a), OS(O)OR^(a), OS(O)₂OR^(a), OR^(b),N(R^(b))R^(c), ⁺N(R^(b))(R^(c))R^(d), P(O)(OR^(a))(OR^(a′)),OP(O)(OR^(a))(OR^(a′)), SiR^(a)R^(a′)R^(a″), C(O)R^(a), C(O)OR^(a),C(O)N(R^(a))R^(a′), OC(O)R^(a), OC(O)OR^(a), OC(O)N(R^(a))R^(a′),N(R^(a))C(O)R^(a′), N(R^(a))C(O)OR^(a′), andN(R^(a))C(O)N(R^(a′))R^(a″), wherein R^(a), R^(a′), and R^(a″) areindependently selected from H and optionally substituted C₁₋₃ alkyl orC₁₋₃ heteroalkyl, and R^(b), R^(c), and R^(d) are independently selectedfrom optionally substituted C₁₋₃ alkyl and C₁₋₃ heteroalkyl;R³, R^(3′), R⁴, and R^(4′) are independently selected from H andoptionally substituted C₁₋₃ alkyl, wherein two or more of R², R³,R^(3′), R⁴, and R^(4′) are optionally joined by one or more bonds toform one or more optionally substituted carbocycles and/or heterocycles,and/or R³+R^(3′) and/or R⁴+R^(4′) are independently ═O, ═S, ═NOR¹⁸, or═NR¹⁸, R¹⁸ being selected from H and optionally substituted C₁₋₃ alkyl;X² is selected from O, C(R¹⁴)(R^(4′)), and NR^(14′), wherein R¹⁴ isselected from H and optionally substituted C₁₋₈ alkyl or C₁₋₈ acyl andwherein R^(14′) is absent or is selected from H and optionallysubstituted C₁₋₈ alkyl or C₁₋₈ acyl;R⁵ and R^(5′) are independently selected from H, OH, SH, NH₂, N₃, NO₂,NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH, halogen, R^(e), SR^(e), S(O)R^(e),S(O)₂R^(e), S(O)OR^(e), S(O)₂OR^(e), OS(O)R^(e), OS(O)₂R^(e),OS(O)OR^(e), OS(O)₂OR^(e), OR^(e), NHR^(e), N(R^(e))R^(f),⁺N(R^(e))(R^(f))R^(g), P(O)(OR^(e))(OR^(f)), OP(O)(OR^(e))(OR^(f)),SiR^(e)R^(f)R^(g), C(O)R^(e), C(O)OR^(e), C(O)N(R^(e))R^(f), OC(O)R^(e),OC(O)OR^(e), OC(O)N(R^(e))R^(f), N(R^(e))C(O)R^(f), N(R^(e))C(O)OR^(f),and N(R^(e))C(O)N(R^(f))R^(g), wherein R^(e), R^(f), and R^(g) areindependently selected from H and optionally substituted C₁₋₃ alkyl,C₁₋₃ heteroalkyl, C₃ cycloalkyl, or C₁₋₃ heterocycloalkyl, two or moreof R^(e), R^(f), and R^(g) optionally being joined by one or more bondsto form one or more optionally substituted carbocycles and/orheterocycles, and R⁶, R^(6′), R⁷, and R^(7′) are independently selectedfrom H, OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH,halogen, R^(e), SR^(e′), S(O)R^(e′), S(O)₂R^(e′), S(O)OR^(e′),S(O)₂OR^(e′), OS(O)R^(e′), OS(O)₂R^(e′), OS(O)OR^(e′), OS(O)₂OR^(e′),OR^(e′), NHR^(e′), N(R^(e′))R^(f′), ⁺N(R^(e′))(R^(f′))R^(g′),P(O)(OR^(e′))(OR^(f′)), OP(O)(OR^(e′))(OR^(f′)), SiR^(e′)R^(f′)R^(g′),C(O)R^(e′), C(O)OR^(e′), C(O)N(R^(e′))R^(f′), OC(O)R^(e′), OC(O)OR^(e′),OC(O)N(R^(e′))R^(f′), N(R^(e′))C(O)R^(f′), N(R^(e′))C(O)OR^(f′), andN(R^(e′))C(O)N(R^(f′))R^(g′), wherein R^(e′), R^(f), and R^(g′) areindependently selected from H and optionally substituted C₁₋₆ alkyl,C₁₋₆ heteroalkyl, C₃₋₇ cycloalkyl, C₁₋₇ heterocycloalkyl, C₆₋₁₂ aryl, orC₁₋₁₂ heteroaryl, two or more of R^(e′), R^(f′), and R^(g′) optionallybeing joined by one or more bonds to form one or more optionallysubstituted carbocycles and/or heterocycles, and/or R⁵+R^(5′), and/orR⁶+R^(6′), and/or R⁷+R^(7′) are independently ═O, ═S, ═NOR¹², or ═NR¹²,R¹² being selected from H and optionally substituted C₁₋₃ alkyl, and/orR^(5′)+R^(6′), and/or R^(6′)+R^(7′), and/or R^(7′)+R^(14′) are absent,which means that a double bond is present between the atoms bearingR^(5′) and R^(6′), and/or R^(6′) and R^(7′), and/or R⁷′ and R^(14′),respectively, two or more of R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′), R¹⁴,and R^(14′) optionally being joined by one or more bonds to form one ormore optionally substituted carbocycles and/or heterocycles;X¹ is selected from O, S, and NR¹³, wherein R¹³ is selected from H andoptionally substituted C₁₋₈ alkyl;X³ is selected from O, S, and NR¹⁵, wherein R¹⁵ is selected from H andoptionally substituted C₁₋₈ alkyl or C₁₋₈ acyl,or —X³— represents —X^(3a) and X^(3b)— wherein X^(3a) is connected tothe carbon to which X⁴ is attached and X^(3b) is connected to the phenylring ortho to R¹⁰, wherein X^(3a) is independently selected from H andoptionally substituted C₁₋₈ alkyl or C₁₋₈ acyl, and X^(3b) is selectedfrom the same pool of substituents as R⁸;

X⁴ is selected from N and CR¹⁶, wherein R¹⁶ is selected from H andoptionally substituted C₁₋₈ alkyl or C₁₋₈ acyl;

X⁵ is selected from O, S, and NR¹⁷, wherein R¹⁷ is selected from H andoptionally substituted C₁₋₈ alkyl or C₁₋₈ acyl;

R⁸, R⁹, R¹⁰, and R¹¹ are each independently selected from H, OH, SH,NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH, halogen, R^(h),SR^(h), S(O)R^(h), S(O)₂R^(h), S(O)OR^(h), S(O)₂OR^(h), OS(O)R^(h),OS(O)₂R^(h), OS(O)OR^(h), OS(O)₂OR^(h), OR^(h), NHR^(h), N(R^(h))R^(i),⁺N(R^(h))(R^(i))R^(j), P(O)(OR^(h))(OR^(i)), OP(O)(OR^(h))(OR^(i)),SiR^(h)R^(i)R^(j), C(O)R^(h), C(O)OR^(h), C(O)N(R^(h))R^(i), OC(O)R^(h),OC(O)OR^(h), OC(O)N(R^(h))R^(i), N(R^(h))C(O)R^(i), N(R^(h))C(O)OR^(i),N(R^(h))C(O)N(R^(i))R^(j), and a water-soluble group, wherein R^(h),R^(i), and R^(j) are independently selected from H and optionallysubstituted C₁₋₁₅ alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅, cycloalkyl, C₁₋₁₅heterocycloalkyl, C₆₋₁₅ aryl, or C₁₋₁₅ heteroaryl, one or more of theoptional substituents in R^(h), R^(i), and/or R^(j) optionally being awater-soluble group, and two or more of R^(h), R^(i), and R^(j)optionally being joined by one or more bonds to form one or moreoptionally substituted carbocycles and/or heterocycles, two or more ofR⁸, R⁹, R¹⁰, R¹¹, and X^(3b) optionally being joined by one or morebonds to form one or more optionally substituted carbocycles and/orheterocycles;

-   -   a and b are independently selected from 0 and 1;    -   c is selected from 0 and 1;    -   provided that in a compound of formula (I):

-   a) at least one of R², R³, R^(3′), R⁴, R^(4′), R⁵, R^(5′), R⁶, and    R^(6′) present is not hydrogen, and

-   b) when R², R³, R^(3′), R⁴, R^(4′), R⁵, and R^(5′) present are    hydrogen, the atom that connects R⁶ or R^(6′) to the remainder of    the compound contains at least two substituents other than hydrogen    or fluorine that are each connected via a single bond to said atom,    and

-   c) when R³, R^(3′), R⁴, R^(4′), R⁵, and R^(5′) present are hydrogen,    R² is not methyl.

In another aspect, the present invention relates to a conjugate of acompound of formula (I) or (II).

In yet another aspect, this invention relates to a compound of formula(III):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinV² is either absent or a functional moiety;each L² is independently absent or a linking group linking V² to L;each L is independently absent or a linking group linking L² to one ormore V¹ and/or Y;each V¹ is independently absent or a conditionally-cleavable orconditionally-transformable moiety, which can be cleaved or transformedby a chemical, photochemical, physical, biological, or enzymaticprocess;each Y is independently absent or a self-eliminating spacer system whichis comprised of 1 or more self-elimination spacers and is linked to V¹,optionally L, and one or more Z;each p and q are numbers representing a degree of branching and are eachindependently a positive integer;z is a positive integer equal to or smaller than the total number ofattachment sites for Z;each Z is independently a compound of formula (I) or (II) as definedhereinabove wherein one or more of X¹, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ mayoptionally in addition be substituted by a substituent of formula (V):

wherein each V^(2′), L², L′, V^(1′), Y′, Z′, p′, q′, and z′ has the samemeaning as defined for V², L², L, V¹, Y, Z, p, q, and z, respectively,the one or more substituents of formula (V) being independentlyconnected to one or more of X¹, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ via Y′,each Z being independently connected to Y through either X¹ or an atomin R⁶, R⁷, R⁸, R⁹, R¹⁰, or R¹¹.

It is noted that z does not represent a degree of polymerization; hencez does not indicate that a number of moieties Z are connected to oneanother.

The present invention also relates to a compound of formula (IV):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinRM is a reactive moiety and L, V¹, Y, Z, p; and z are as defined above,except that L is now linking RM to one or more V¹ and/or Y, and V¹, Y,and Z may contain protecting groups, and the one or more V^(2′)-L^(2′)moieties optionally present in Z as defined hereinabove may optionallyand independently be replaced by RM′, which is a reactive moiety, andwherein, if there is more than 1 reactive moiety in (IV), some or allreactive moieties are the same or different. These linker-agentconjugates of formula (IV) may or may not be considered intermediatesfor compounds of formula (III).

Furthermore, this invention relates to the cyclopropyl ring-containinganalogs of compounds of formulae (I) and (II), which are formed throughrearrangement of and concomitant elimination of H—R¹ from thecorresponding seco compounds of formulae (I) and (II) (FIG. 1). Saidcyclopropyl ring-containing analogs are believed to be the activespecies, allegedly being formed from compounds of formulae (I) and (II)in vivo via said rearrangement.

This invention relates to enantiomerically pure and/ordiastereomerically pure compounds of formulae (I)-(IV) as well as toenantiomeric and/or diastereomeric mixtures of compounds of formulae(I)-(IV).

Compounds of formulae (I) and (II) were unexpectedly found to exhibit ahigh in vitro cytotoxicity. This makes these compounds suitable forapplication in drug delivery purposes, including drug targeting andcontrolled release applications using compounds of formulae (III) and/or(IV).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the rearrangement of a seco compound to acyclopropyl-containing compound.

FIG. 2 illustrates the preparation of agent 4 from compound 1.

FIG. 3 depicts the synthesis of agent 7 from compound 5.

FIG. 4 shows the preparation of agent 9 from compound 5.

FIG. 5 illustrates the synthesis of agent 11 from compound 10.

FIG. 6 depicts the preparation of agent 12 from compound 10.

FIG. 7 provides some exemplary structures of linker-agent conjugates.

DESCRIPTION OF THE INVENTION

The following detailed description is provided so that the invention maybe more fully understood.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art.

The term “antibody”, as used herein, refers to a full lengthimmunoglobulin molecule, an immunologically active portion of afull-length immunoglobulin molecule, or a derivative of a full lengthimmunoglobulin molecule or an active portion thereof, i.e., a moleculethat contains an antigen-binding site that immunospecifically binds anantigen of a target of interest or part thereof, such targets including,but not limited to, tumor cells. The immunoglobulin can be of any type(e.g., IgG, IgE, IgM, IgD, IgA, or IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1, or IgA2), or subclass of immunoglobulin molecule. Theimmunoglobulin can be derived from any species, e.g., human, rodent(e.g., mouse, rat, or hamster), donkey, sheep, rabbit, goat, guinea pig,camelid, horse, cow, or chicken, but preferably, it is of human, murine,or rabbit origin. Antibodies useful in the invention include, but arenot limited to, monoclonal, polyclonal, bispecific, human, humanized, orchimeric antibodies, single chain antibodies, Fv fragments, Fabfragments, F(ab′) fragments, F(ab′)₂ fragments, fragments produced by aFab expression library, anti-idiotypic antibodies, CDRs, andepitope-binding fragments of any of the above that immunospecificallybind to an antigen-of-interest.

The term “leaving group” refers to a group that can be substituted byanother group in a nucleophilic substitution reaction. Such leavinggroups are well-known in the art, and examples include, but are notlimited to, a halide (fluoride, chloride, bromide, and iodide), asulfonate (e.g., an optionally substituted C₁₋₆ alkanesulfonate, such asmethanesulfonate and trifluoromethanesulfonate, and an optionallysubstituted C₇₋₁₂ alkylbenzenesulfonate, such as p-toluenesulfonate),succinimide-N-oxide, p-nitrophenoxide, pentafluorophenoxide,tetrafluorophenoxide, a carboxylate, and an alkoxycarboxylate. Forsubstitutions at saturated carbon, halides and sulfonates are preferredleaving groups.

The term “water-soluble group” refers to a functional group that is wellsolvated in aqueous environments and that imparts improved watersolubility to the compound to which it is attached. Examples ofwater-soluble groups include, but are not limited to, polyalcohols,straight chain or cyclic saccharides, primary, secondary, tertiary, orquaternary amines and polyamines, sulfate groups, carboxylate groups,phosphate groups, phosphonate groups, ascorbate groups, glycols,including polyethylene glycols, and polyethers.

The term “substituted”, when used as an adjective to “alkyl”,“heteroalkyl”, “cycloalkyl”, “heterocycloalkyl”, “aryl”, “heteroaryl”,and the like, indicates that said “alkyl”, “heteroalkyl”, “cycloalkyl”,“heterocycloalkyl”, “aryl”, or “heteroaryl” group contains one or moresubstituents (not being hydrogen). Exemplary substituents include, butare not limited to, OH, ═O, ═S, ═NR^(k), ═N—OR^(k), SH, NH₂, NO₂, NO,N₃, CF₃, CN, OCN, SCN, NCO, NCS, C(O)NH₂, C(O)H, C(O)OH, halogen, R^(k),SR^(k), S(O)R^(k), S(O)OR^(k), S(O)₂R^(k), S(O)₂OR^(k), OS(O)R^(k),OS(O)OR^(k), OS(O)₂R^(k), OS(O)₂OR^(k), OP(O)(OR^(k))(OR^(l)),P(O)(OR^(k))(OR^(l)), OR^(k), NHR^(k), N(R^(k))R^(l),⁺N(R^(k))(R^(l))R^(m), Si(R^(k))(R^(l))(R^(m)), C(O)R^(k), C(O)OR^(k),C(O)N(R^(k))R^(l), OC(O)R^(k), OC(O)OR^(k), OC(O)N(R^(k))R^(l),N(R^(k))C(O)R^(l), N(R^(k))C(O)OR^(l), N(R^(k))C(O)N(R^(l))R^(m), andthe thio derivatives of these substituents, or a protonated ordeprotonated form of any of these substituents, wherein R^(k), R^(l),and R^(m) are independently selected from H and optionally substitutedC₁₋₁₅ alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅ cycloalkyl, C₁₋₅ heterocycloalkyl,C₆₋₁₅ aryl, or C₁₋₅ heteroaryl, or a combination thereof, two or more ofR^(k), R^(l), and R^(m) optionally being joined by one or more bonds toform one or more optionally substituted carbocycles and/or heterocycles.

The term “aryl” as used herein refers to a carbocyclic aromaticsubstituent comprising 6-24 carbon atoms, which may consist of one ringor two or more rings fused together. Examples of aryl groups include,but are not limited to, phenyl, naphthyl, and anthracenyl.

The term “heteroaryl” as used herein refers to a carbocyclic aromaticsubstituent comprising 1 to 24 carbon atoms and at least one heteroatomselected from the group consisting of oxygen, nitrogen, sulfur, silicon,and phosphorus, which may consist of one ring or two or more rings fusedtogether. Examples of heteroaryl groups include, but are not limited to,pyridinyl, furanyl, pyrrolyl, triazolyl, pyrazolyl, imidazolyl,thiophenyl, indolyl, benzofuranyl, benzimidazolyl, indazolyl,benzotriazolyl, benzisoxazolyl, and quinolinyl. In one embodiment, aheteroaryl group comprises 1 to 4 heteroatoms. It should be noted that“C₁ heteroaryl group” denotes that there is only one carbon present inthe ring system of the heteroaromatic group. An example of such aheteroaromatic group is a tetrazolyl group.

The term “alkyl” as used herein refers to a straight chain or branched,saturated or unsaturated hydrocarbyl substituent. Examples of alkylgroups include, but are not limited to, methyl, ethyl, propyl, butyl,pentyl, hexyl, octyl, decyl, isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, 2-methylbutyl, vinyl, allyl, 1-butenyl, 2-butenyl,isobutylenyl, 1-pentenyl, 2-pentenyl, and 1-butynyl.

The term “heteroalkyl” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbyl substituent in which atleast one carbon atom is replaced by a heteroatom. Examples include, butare not limited to, methyloxymethyl, ethyloxymethyl, methyloxyethyl,ethyloxyethyl, methylaminomethyl, dimethylaminomethyl, methylaminoethyl,dimethylaminoethyl, methylthiomethyl, ethylthiomethyl, ethylthioethyl,and methylthioethyl.

The term “cycloalkyl” as used herein refers to a saturated orunsaturated non-aromatic cyclic hydrocarbyl substituent, which mayconsist of one ring or two or more rings fused together. Examplesinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl,1,3-cyclohexadienyl, decalinyl, and 1,4-cyclohexadienyl.

The term “heterocycloalkyl” as used herein refers to a saturated orunsaturated non-aromatic cyclic hydrocarbyl substituent, which mayconsist of one ring or two or more rings fused together, wherein atleast one carbon in one of the rings is replaced by a heteroatom.Examples include, but are not limited to, tetrahydrofuranyl,pyrrolidinyl, piperidinyl, 1,4-dioxanyl, decahydroquinolinyl,piperazinyl, oxazolidinyl, and morpholinyl. It should be noted that “C₁heterocycloalkyl group” denotes that there is only one carbon present inthe ring system of the heterocycloalkane. An example of such a group isa dioxiranyl group.

The number of carbon atoms that an “alkyl”, “heteroalkyl”, “cycloalkyl”,“heterocycloalkyl”, “aryl”, “heteroaryl”, and the like, may contain isindicated by a designation preceding said terms (i.e., C₁₋₁₀ alkyl meansthat said alkyl may contain from one to ten carbons).

The term “carbocycle” herein refers to a saturated or unsaturatedcycloalkane or arene moiety, wherein the terms “cycloalkane” and “arene”are defined as parent moieties of the “cycloalkyl” and “aryl”substituents, respectively, as defined hereinabove.

The term “heterocycle” herein refers to a saturated or unsaturatedheterocycloalkane or heteroarene moiety, wherein the terms“heterocycloalkane” and “heteroarene” are defined as parent moieties ofthe “heterocycloalkyl” and “heteroaryl” substituents, respectively, asdefined hereinabove.

The extension “-ylene” as opposed to “-yl” in for example “alkylene” asopposed to “alkyl” indicates that said for example “alkylene” is adivalent moiety connected to one or two other moieties via one doublebond or two covalent single bonds, respectively, as opposed to being amonovalent group connected to one moiety via one covalent single bond insaid for example “alkyl”. The term “alkylene” therefore refers to astraight chain or branched, saturated or unsaturated hydrocarbylenemoiety; the term “heteroalkylene” as used herein refers to a straightchain or branched, saturated or unsaturated hydrocarbylene moiety inwhich at least one carbon is replaced by a heteroatom; the term“arylene” as used herein refers to a carbocyclic aromatic moiety, whichmay consist of one ring or two or more rings fused together; the term“heteroarylene” as used herein refers to a carbocyclic aromatic moiety,which may consist of one ring or two or more rings fused together,wherein at least one carbon in one of the rings is replaced by aheteroatom; the term “cycloalkylene” as used herein refers to asaturated or unsaturated non-aromatic cyclic hydrocarbylene moiety,which may consist of one ring or two or more rings fused together; theterm “heterocycloalkylene” as used herein refers to a saturated orunsaturated non-aromatic cyclic hydrocarbylene moiety, which may consistof one ring or two or more rings fused together, wherein at least onecarbon in one of the rings is replaced by a heteroatom. Exemplarydivalent moieties include those examples given for the monovalent groupshereinabove in which one hydrogen atom is removed.

The prefix “poly” in “polyalkylene”, “polyheteroalkylene”,“polyarylene”, “polyheteroarylene”, polycycloalkylene”,“polyheterocycloalkylene”, and the like, indicates that two or more ofsuch “-ylene” moieties, e.g., alkylene moieties, are joined together toform a branched or unbranched multivalent moiety containing two or moreattachment sites for adjacent moieties.

Certain compounds of the invention possess chiral centers and/or doublebonds, and/or may have tautomers; the tautomeric, enantiomeric,diastereomeric, and geometric mixtures of two or more isomers, in anycomposition, as well as the individual isomers (including tautomers) areencompassed within the scope of the present invention. Whenever the term“isomer” is used, it refers to a tautomeric, enantiomeric,diastereomeric, and/or geometric isomer or to a mixture of two or moreof these isomers, unless the context dictates otherwise.

The term “bond” herein refers to a covalent connection between two atomsand may refer to a single bond, a double bond, or a triple bond, or, ifresonance structures are possible, the bond order of said bond may bedifferent in two or more of these resonance structures. For example, ifthe bond is part of an aromatic ring, the bond may be a single bond inone resonance structure and a double bond in another resonancestructure.

The compounds of the invention may also contain unnatural proportions ofatomic isotopes at one or more atoms that constitute such compounds. Allisotopic variations of the compounds of this invention, whetherradioactive or not, are intended to be encompassed within the scope ofthis invention.

The phrase “pharmaceutically active salt” as used herein refers to apharmaceutically acceptable organic or inorganic salt of a compound ofthe invention. For compounds containing one or more basic groups, e.g.,an amine group, acid addition salts can be formed. For compoundscontaining one or more acidic groups, e.g., a carboxylic acid group,base addition salts can be formed. For compounds containing both acidicand basic groups, zwitterions may in addition be obtained as salts. Whenthe compound of the invention comprises more than one charged atom orgroup, there may be multiple (distinct) counterions.

The phrase “pharmaceutically acceptable solvate” refers to anassociation of one or more solvent molecules and a compound of theinvention. Examples of solvents that form pharmaceutically acceptablesolvates include, but are not limited to, water, isopropyl alcohol,ethanol, methanol, DMSO, ethyl acetate, and acetic acid. When referringto water as a solvate, the term “hydrate” can be used.

The term “conjugate” hereinbelow refers to a compound of formula (III).

The term “linker-agent conjugate” hereinbelow refers to a compound offormula (IV).

The term “agent” hereinbelow refers to a compound of formula (I), (II),(I′), or (II′).

The term “targeting moiety” refers to any moiety that specifically bindsor reactively associates or complexes with a moiety specifically or inrelative excess present at or near the target site, on, in, or near thetarget cell, or in (the proximity of) the target tissue or organ, e.g.,a receptor, a receptor complex, substrate, antigenic determinant, orother receptive moiety, or that can target the conjugate to the targetsite via other mechanisms by virtue of its nature, e.g., through the EPReffect. Examples of a targeting moiety include, but are not limited to,an aptamer, an antibody or antibody fragment, a polymer, a dendrimer, alectin, a biologic response modifier, an enzyme, a vitamin, a growthfactor, a steroid, a sugar residue, an oligosaccharide residue, acarrier protein, and a hormone, or any combination thereof.

The phrase “moiety that improves the pharmacokinetic properties of thecompound” refers to a moiety that changes the pharmacokinetic propertiesof a compound of this invention in such a way that a better therapeuticeffect can be obtained. The moiety can for example increase the watersolubility, increase the circulation time, or reduce immunogenicity.

The term “linking group” refers to a structural element of a compoundthat links one structural element of said compound to one or more otherstructural elements of said same compound.

The phrase “a number representing degree of branching” is used to denotethat the subscript number next to a closing bracket represents how manyunits of the moiety within the brackets are each directly attached tothe moiety immediately to the left of the corresponding opening bracket.For example, A-(B)_(b) with b being a number representing a degree ofbranching means that b units B are all directly attached to A. Thismeans that when b is 2, the formula reduces to B-A-B.

The phrase “a number representing degree of polymerization” is used todenote that the subscript number next to a closing bracket representshow many units of the moiety within the brackets are connected to eachother. For example, A-(B)_(b) with b being a number representing adegree of polymerization means that when b is 2, the formula reduces toA-B-B.

The term “single-release spacer” refers to a self-elimination spacerthat can release one moiety upon self-immolation.

The term “multiple-release spacer” refers to a self-elimination spacerthat can release two or more moieties upon repetitive self-immolation.

The term “electronic cascade spacer” refers to a self-eliminationspacer, either branched or unbranched, which may self-eliminate throughone or more 1,2+2n electronic cascade eliminations (n≧1).

The term “ω-amino aminocarbonyl cyclization spacer” refers to aself-elimination spacer that may eliminate through a cyclization processunder formation of a cyclic ureum derivative.

The term “spacer system” refers to a single spacer moiety or to two ormore of the same or different spacer moieties coupled together. A spacersystem may be branched or unbranched and contain one or more attachmentsites for Z as well as V¹ and optionally L.

In this document and in its claims, the verbs “to comprise”, “to have”,“to contain” and their conjugations are used in their non-limiting senseto mean that items that are “comprised”, “had”, or “contained” areincluded, but items non-specifically mentioned are not excluded. Inaddition, reference to an element by the indefinite article “a” or “an”does not exclude the possibility that more than one of the element ispresent; unless the context clearly requires that there be one and onlyone of the elements. The indefinite article “a” or “an” thus usuallymeans “at least one”.

In the generic structures throughout this description and in the claimsletters are used to define structural elements. Some of these letterscan be mistaken to represent an atom, such as C, N, O, P, K, B, F, S, U,V, W, I, and Y. To avoid confusion whenever these letters do notrepresent an atom they are given in bold typeface.

When there are one or more adjectives and/or adjective phrases to a nounthat is a) the first in a list of nouns or b) that is anywhere in themiddle of a list of nouns and said noun and adjectives together arepreceded by the word “and” or “or”, the adjectives do not only bear onsaid noun, but on all following nouns separately, unless the contextdictates otherwise. This for example means that the phrase “optionallysubstituted C₁₋₄ alkyl, C₁₋₄ heteroalkyl, C₃₋₇ cycloalkyl, or C₁₋₇heterocycloalkyl” should be read as “optionally substituted C₁₋₄ alkyl,optionally substituted C₁₋₄ heteroalkyl, optionally substituted C₃₋₇cycloalkyl, or optionally substituted C₁₋₇ heterocycloalkyl” and thatthe phrase “C₁₋₄ alkyl, C₁₋₄ heteroalkyl, and optionally substitutedC₃₋₇ cycloalkyl, C₆₋₈ aryl, or C₁₋₇ heterocycloalkyl” should be read as“C₁₋₄ alkyl, C₁₋₄ heteroalkyl, and optionally substituted C₃₋₇cycloalkyl, optionally substituted C₆₋₈ aryl, or optionally substitutedC₁₋₇ heterocycloalkyl”.

Throughout this description and in the claims molecular structures orparts thereof are drawn. As usual in such drawings bonds between atomsare represented by lines, in some cases, to indicate stereochemistry, bybold or broken or wedged lines. Usually a line ending in space (a“loose” end), i.e., at one end not having another line or specific atomconnected to it, represents a CH₃ group.

This is correct for the drawings representing the compounds of thisinvention. For those structures representing a structural element of thecompounds of this invention a line ending in space may indicate theposition of attachment of another structural element of the compound.This has been indicated with a wavy line perpendicular to and crossingthe “loose” line in most drawings.

Furthermore, the structures or parts thereof have been drawn, under theassumption that the structures are read from left to right, meaning thatfor example in the drawings of compounds of formula (III) V² (ifpresent) is always located on the left side and Z is always located onthe right side of such structures.

The following abbreviations are used herein and have the indicateddefinitions: AIBN: 2,2′-azobis(2-methylpropionitril); DMF:N,N-dimethylformamide; EDC:1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; EtOAc: ethyl acetate;THF: tetrahydrofuran; TTMSS: tris(trimethylsilyl)silane.

Agents, Linker-Agent Conjugates, and Conjugates

The present invention provides novel agents that belong to the class ofthe DNA-alkylating agents CC-1065 and the duocarmycins. Furthermore, theinvention relates to novel conjugates of these agents and tolinker-agent conjugates, which may or may not serve as intermediates forsaid conjugates.

The agents of the present invention are deemed to be used to treat anillness that is characterized by undesired (cell) proliferation. Forexample, an agent of this invention can be used to treat a tumor,cancer, an autoimmune disease, or an infectious disease.

The conjugates of the present invention are in one aspect deemed to beapplicable to target agents of formulae (I) and (II) to a specifictarget site where the conjugate can be converted into one or more agentsor be induced to be converted into one or more of said agents. Thisinvention can furthermore find application in (non-specific) controlledrelease of one or more of said agents from a conjugate, with the aim offor example enhancing pharmacokinetic properties.

Compounds of formulae (I) and (II) were unexpectedly found to exhibit ahigh in vitro cytotoxicity, which makes these compounds suitable forapplication in drug delivery purposes. An explanation for the high invitro cytotoxicity may be steric shielding of the alkylating site in acompound of formula (I) or (II).

Agents

In one aspect, the present invention provides a compound of formula (I)or (II):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinR¹ is a leaving group;R² is selected from H, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH,halogen, R^(b), SR^(b), S(O)R^(a), S(O)₂R^(a), S(O)OR^(a), S(O)₂OR^(a),OS(O)R^(a), OS(O)₂R^(a), OS(O)OR^(a), OS(O)₂OR^(a), OR^(b),N(R^(b))R^(c), ⁺N(R^(b))(R^(c))R^(d), P(O)(OR^(a))(OR^(a′)),OP(O)(OR^(a))(OR^(a′)), SiR^(a)R^(a′)R^(a″), C(O)R^(a), C(O)OR^(a),C(O)N(R^(a))R^(a′), OC(O)R^(a), OC(O)OR^(a), OC(O)N(R^(a))R^(a′),N(R^(a))C(O)R^(a′), N(R^(a))C(O)OR^(a′), andN(R^(a))C(O)N(R^(a′))R^(a′), wherein R^(a), R^(a′), and R^(a″) areindependently selected from H and optionally substituted C₁₋₃ alkyl orC₁₋₃ heteroalkyl, and R^(b), R^(c), and R^(d) are independently selectedfrom optionally substituted C₁₋₃ alkyl and C₁₋₃ heteroalkyl;R³, R^(3′), R⁴, and R^(4′) are independently selected from H andoptionally substituted C₁₋₃ alkyl, wherein two or more of R², R³,R^(3′), R⁴, and R^(4′) are optionally joined by one or more bonds toform one or more optionally substituted carbocycles and/or heterocycles,and/or R³+R^(3′) and/or R⁴+R^(4′) are independently ═O, ═S, ═NOR¹⁸, or═NR¹⁸, R¹⁸ being selected from H and optionally substituted C₁₋₃ alkyl;X² is selected from O, C(R¹⁴)(R^(14′)), and NR^(14′), wherein R¹⁴ isselected from H and optionally substituted C₁₋₈ alkyl or C₁₋₈ acyl andwherein R^(14′) is absent or is selected from H and optionallysubstituted C₁₋₈ alkyl or C₁₋₈ acyl;R⁵ and R^(5′) are independently selected from H, OH, SH, NH₂, N₃, NO₂,NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH, halogen, R^(e), SR^(e), S(O)R^(e),S(O)₂R^(e), S(O)OR^(e), S(O)₂OR^(e), OS(O)R^(e), OS(O)₂R^(e),OS(O)OR^(e), OS(O)₂OR^(e), OR^(e), NHR^(e), N(R^(e))R^(f),⁺N(R^(e))(R^(f))R^(g), P(O)(OR^(e))(OR^(f)), OP(O)(OR^(e))(OR^(f)),SiR^(e)R^(f)R^(g), C(O)R^(e), C(O)OR^(e), C(O)N(R^(e))R^(f), OC(O)R^(e),OC(O)OR^(e), OC(O)N(R^(e))R^(f), N(R^(e))C(O)R^(f), N(R^(e))C(O)OR^(f),and N(R^(e))C(O)N(R^(e))R^(g), wherein R^(e), R^(f), and R^(g) areindependently selected from H and optionally substituted C₁₋₃ alkyl,C₁₋₃ heteroalkyl, C₃ cycloalkyl, or C₁₋₃ heterocycloalkyl, two or moreof R^(e), R^(f), and R^(g) optionally being joined by one or more bondsto form one or more optionally substituted carbocycles and/orhetcrocycles, and R⁶, R^(6′), R⁷, and R^(7′) are independently selectedfrom H, OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH,halogen, R^(e′)SR^(e′,) S(O)R^(e′), S(O)₂R^(e′), S(O)OR^(e′),S(O)₂OR^(e′), OS(O)R^(e′), OS(O)₂R^(e), OS(O)OR^(e′), OS(O)₂OR^(e′),OR^(e′,) NHR^(e′,) N(R^(e′))R^(f′), ⁺N(R^(e′))(R^(f′))R^(g′),P(O)(OR^(e′))(OR^(f)), OP(O)(OR^(e′))(OR^(f)), SiR^(e′)R^(f′)R^(g′),C(O)R^(e′,) C(O)OR^(e′), C(O)N(R^(e′))R^(f′), OC(O)R^(e), OC(O)OR^(e′),OC(O)N(R^(e′))R^(f′), N(R^(e′))C(O)R^(f′), N(R^(e′))C(O)OR^(f′), andN(R^(e′))C(O)N(R^(f′))R^(g′), wherein R^(e′), R^(f′), and R^(g′) areindependently selected from H and optionally substituted C₁₋₆ alkyl,C₁₋₆ heteroalkyl, C₃₋₇ cycloalkyl, C₁₋₇ heterocycloalkyl, C₆₋₁₂ aryl, orC₁₋₁₂ heteroaryl, two or more of R^(e′), R^(f′), and R^(g′) optionallybeing joined by one or more bonds to form one or more optionallysubstituted carbocycles and/or heterocycles,and/or R⁵+R^(5′), and/or R⁶+R^(6′), and/or R⁷+R^(7′) are independently═O, ═S, ═NOR¹², or ═NR¹², R¹² being selected from H and optionallysubstituted C₁₋₃ alkyl,and/or R^(5′)+R^(6′), and/or R^(6′)+R^(7′), and/or R^(7′)+R^(14′) areabsent, which means that a double bond is present between the atomsbearing R^(5′) and R^(6′), and/or R^(6′) and R^(7′), and/or R^(7′) andR¹⁴, respectively, two or more of R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′),R¹⁴, and R^(14′) optionally being joined by one or more bonds to formone or more optionally substituted carbocycles and/or heterocycles;X¹ is selected from O, S, and NR¹³, wherein R¹³ is selected from H andoptionally substituted C₁₋₈ alkyl;X³ is selected from O, S, and NR¹⁵, wherein R¹⁵ is selected from H andoptionally substituted C₁₋₈ alkyl or C₁₋₈ acyl,or —X³— represents —X^(3a) and X^(3b)— wherein X^(3a) is connected tothe carbon to which X⁴ is attached and X^(3b) is connected to the phenylring ortho to R¹⁰, wherein X^(3a) is independently selected from H andoptionally substituted C₁₋₈ alkyl or C₁₋₈ acyl, and X^(3b) is selectedfrom the same pool of substituents as R⁸;X⁴ is selected from N and CR¹⁶, wherein R¹⁶ is selected from H andoptionally substituted C₁₋₈ alkyl or C₁₋₈ acyl;X⁵ is selected from O, S, and NR¹⁷, wherein R¹⁷ is selected from H andoptionally substituted C₁₋₈ alkyl or C₁₋₈ acyl;R⁸, R⁹, R¹⁰, and R¹¹ are each independently selected from H, OH, SH,NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH, halogen, R^(h),SR^(h), S(O)R^(h), S(O)₂R^(h), S(O)OR^(h), S(O)₂OR^(h), OS(O)R^(h),OS(O)₂R^(h), OS(O)OR^(h), OS(O)₂OR^(h), OR^(h), NHR^(h), N(R^(h))R^(i),⁺N(R^(h))(R^(i))R^(j), P(O)(OR^(h))(OR^(i)), OP(O)(OR^(h))(OR^(i)),SiR^(h)R^(i)R^(j), C(O)R^(h), C(O)OR^(h), C(O)N(R^(h))R^(i), OC(O)R^(h),OC(O)OR^(h), OC(O)N(R^(h))R^(i), N(R^(h))C(O)R^(i), N(R^(h))C(O)OR^(i),N(R^(h))C(O)N(R^(i))R^(j), and a water-soluble group, wherein R^(h),R^(i), and R^(j) are independently selected from H and optionallysubstituted C₁₋₁₅ alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅ cycloalkyl, C₁₋₁₅heterocycloalkyl, C₆₋₁₅ aryl, or C₁₋₁₅ heteroaryl, one or more of theoptional substituents in R^(h), R^(i), and/or R^(j) optionally being awater-soluble group, and two or more of R^(h), R^(i), and R^(j)optionally being joined by one or more bonds to form one or moreoptionally substituted carbocycles and/or heterocycles, two or more ofR⁸, R⁹, R¹⁰, R¹¹, and X^(3b) optionally being joined by one or morebonds to form one or more optionally substituted carbocycles and/orheterocycles;a and b are independently selected from 0 and 1;c is selected from 0 and 1;provided that in a compound of formula (I):

-   a) at least one of R², R³, R^(3′), R⁴, R^(4′), R⁵, R^(5′), R⁶, and    R^(6′) present is not hydrogen, and-   b) when R², R³, R^(3′), R⁴, R^(4′), R⁵, and R^(5′) present are    hydrogen, the atom that connects R⁶ or R^(6′) to the remainder of    the compound contains at least two substituents other than hydrogen    or fluorine that are each connected via a single bond to said atom,    and-   c) when R³, R^(3′), R⁴, R^(4′), R⁵, and R^(5′) present are hydrogen,    R² is not methyl.

In a further aspect, this invention relates to a compound of formula(I′) or (II′):

wherein all substituents have the same meaning as described forcompounds of formulae (I) and (II) above. Compounds of formulae (I) and(II) are alleged to be converted to (I′) and (II′), respectively, invivo with concomitant elimination of H—R¹, as schematically illustratedin FIG. 1 for a compound of formula (I).

Therefore, this invention relates to a compound of formula (I′) or(II′), said compound comprising a cyclopropyl group, which can be formedthrough rearrangement of and concomitant elimination of H—R¹ from acompound of formula (I) or (II).

It should be understood that in this entire document, when referring toa compound of formula (I) or (II), this includes reference to a compoundof formula (I′) or (II′), respectively, unless structural parts of (I)and (II) not present in (I′) and (II′) are concerned or the contextdictates otherwise. Similarly, when referring to a structural part(fragment), linker-agent conjugate, or conjugate of formula (I) or (II),this includes reference to a similar structural part (fragment),linker-agent conjugate, or conjugate of formula (I′) or (II′),respectively, unless structural parts of (I) and (II) not present in(I′) and (II′) are concerned or the context dictates otherwise.

It should also be understood that when reference is made to a compoundof formula (I) or (II) and the scope of R² is specified, thisspecification only affects a compound of formula (I) as R² is absent ina compound of formula (II). Therefore, wherever it reads “R²” in thisdocument, one could read “R² (if present)”.

It should further be understood that this invention relates toenantiomerically pure and/or diastereomerically pure compounds offormulae (I) and (II) as well as to enantiomeric and/or diastereomericmixtures of compounds of formulae (I) and (II).

Considerations about substituent effects in compounds of formulae (I)and (II) and their cyclopropyl-containing analogs given in this documentare presented without consenting to a specific mechanism of action forcompounds of formulae (I) and (II) and their cyclopropyl-containinganalogs.

Compounds of formula (I) and (II) can be considered to be built up of aDNA-binding unit and a DNA-alkylating unit, as indicated in the figuresabove.

The DNA-alkylating unit of compounds of formulae (I) and (II) isconsidered to contain the site of alkylation. Alkylation of DNA mayoccur through attack of DNA on the carbon bearing R¹ in a compound offormula (I) or (II) or on that same carbon in the cyclopropyl-containinganalog of said compound.

In one embodiment, this invention relates to a compound of formula (I).In another embodiment, this invention relates to a compound of formula(II).

R¹ in a compound of formula (I) or (II) is a leaving group.

In one embodiment, the leaving group R¹ is selected from halogen, azide(N₃), carboxylate [OC(O)R^(n)], carbonate [OC(O)OR^(n)], carbamate[OC(O)N(R^(n))R^(n′)], and OS(O)₂R^(o), wherein R^(n) and R^(n′) areindependently selected from optionally substituted C₁₋₆ alkyl and C₁₋₆heteroalkyl and wherein R^(o) is selected from C₁₋₆ perhaloalkyl andoptionally substituted C₁₋₆ alkyl, benzyl, or phenyl. In one embodiment,R¹ is selected from halogen and OS(O)₂R^(o). In another embodiment, theleaving group R¹ in a compound of formula (I) or (II) is a halogen. Inanother embodiment, R¹ is selected from chloride (Cl), bromide (Br), andiodide (I). In yet another embodiment, R¹ is chloride (Cl). In yetanother embodiment, R¹ is bromide (Br). In yet another embodiment, R¹ isOS(O)₂R^(o) In yet another embodiment, R¹ is selected from triflate[OS(O)₂CF₃], tosylate [OS(O)₂C₆H₄CH₃], and mesylate [OS(O)₂CH₃].

By varying the leaving group R¹, one may tune the alkylating activity ofthe seco agents and affect the transformation rate of a seco agent to acyclopropyl-containing agent of formula (I′) or (II′). If the leavingcapability of R¹ is too good, this may cause the seco agent to become an(aspecific) alkylating agent as well, which may decrease thecytotoxicity quotient of conjugates of compounds of formulae (I) and(II) as the agent may be able to alkylate while still being bound in theconjugate. On the other hand, if R¹ is too bad a leaving group, the secoagent may not close to form a cyclopropyl-containing agent, believed tobe the active species, which may reduce its cytotoxicity and, mostlikely, reduce the cytotoxicity quotient. Therefore, in one embodiment,the Swain-Scott parameter s of the alkylating site is larger than 0.3.In other embodiments, the Swain-Scott parameter s is larger than 0.5 or0.7 or 1.0.

The size of R¹ may affect the non-DNA alkylation rate of a compound offormula (I) or (II) or a conjugate thereof. If R¹ is a relatively bulkygroup, aspecific alkylation may be reduced as the carbon bearing R¹ issomewhat shielded.

Another means to tune the alkylating activity of the seco agents andtheir cyclopropyl-containing derivatives may be to somewhat shield thecarbon to which the leaving group R¹ is attached or on whichnucleophilic attack can occur by choosing at least one of R², R³,R^(3′), R⁴, R^(4′), R⁵, R^(5′), R⁶, and R^(6′) present to be other thanhydrogen. Shielding of said carbon may reduce aspecific alkylation bycompounds of formulae (I) and (II) and their cyclopropyl-containinganalogs and by their conjugates as well. Although introduction of sterichindrance may also affect the DNA alkylation rate, it may be reasonableto assume that aspecific alkylation may be affected relatively more thanDNA alkylation as the latter occurs presumably after the agent isideally positioned for nucleophilic attack being bound to the DNA minorgroove. The carbon bearing R¹ in a compound of formula (II), being asecondary carbon atom, is already somewhat shielded in comparison to thecarbon bearing R¹ in a compound of formula (I) when R² is H. In thisrespect, a compound of formula (II) may be compared to a compound offormula (I) in which R² is other than hydrogen. Further shielding mayhowever be accomplished by choosing one or more of R³, R^(3′), R⁴,R^(4′), R⁵, R^(5′), R⁶, and R^(6′) present to be other than hydrogen.

In one embodiment, R² is hydrogen.

In another embodiment, R² is selected from N₃, NO₂, NO, CF₃, CN,C(O)NH₂, C(O)H, C(O)OH, halogen, R^(b), SR^(b), S(O)R^(a), S(O)₂R^(a),S(O)OR^(a), S(O)₂OR^(a), OS(O)R^(a), OS(O)₂R^(a), OS(O)OR^(a),OS(O)₂OR^(a), OR^(b), N(R^(b))R^(c), ⁺N(R^(b))(R^(c))R^(d),P(O)(OR^(a))(OR^(a′)), OP(O)(OR^(a))(OR^(a′)), SiR^(a)R^(a′)R^(a″),C(O)R^(a), C(O)OR^(a), C(O)N(R^(a))R^(a′), OC(O)R^(a), OC(O)OR^(a),OC(O)N(R^(a))R^(a′), N(R^(a))C(O)R^(a), N(R^(a))C(O)OR^(a), andN(R^(a))C(O)N(R^(a′))R^(a″), wherein R^(a), R^(a′), and R^(a″) areindependently selected from H and optionally substituted C₁₋₃ alkyl orC₁₋₃ heteroalkyl, and R^(b), R^(c), and R^(d) are independently selectedfrom optionally substituted C₁₋₃ alkyl and C₁₋₃ heteroalkyl.

In one embodiment, R² is optionally substituted C₁₋₃ alkyl. In anotherembodiment, R² is selected from methyl, ethyl, propyl, and isopropyl. Inanother embodiment, R² is methyl. In other embodiments, R² is ethyl orpropyl or isopropyl.

In a further embodiment, R² is selected from N₃, halogen, OS(O)₂R^(a),CF₃, NO₂, N(R^(b))R^(c), OR^(b) C(O)R^(a), C(O)OR^(a), OC(O)R^(a),OC(O)OR^(a), OC(O)N(R^(a))R^(a′), and C(O)N(R^(a))R^(a).

In another embodiment, R² is selected from chloride (Cl), bromide (Br),azide (N₃), CF₃, NO₂, methoxy (OMe), and acetyl [C(O)CH₃].

In another embodiment, R² is selected from chloride and bromide. In afurther embodiment, R² is chloride. In another further embodiment, R² isbromide. In other embodiments, R² is azide (N₃) or CF₃ or NO₂ or methoxy(OMe) or acetyl [C(O)CH₃].

R¹ and R² may be equal to each other. In such a case, the synthesis of acompound of formula (I) may be accomplished in a higher yield and/orwith more ease as the compound contains one less chiral center than whensubstituents R¹ and R² are different from each other and R² is nothydrogen.

In one embodiment, R¹ and R² are equal to each other. In anotherembodiment, R¹ and R² are both either bromide or chloride. In anotherembodiment, R¹ and R² are both chloride (Cl). In another embodiment, R¹and R² are both bromide (Br).

Alternatively, or simultaneously, steric shielding of the carbon bearingR¹ may be introduced by choosing one or more of R³, R^(3′), R⁴, andR^(4′) to be other than hydrogen. In one embodiment, R³, R^(3′), R⁴, andR^(4′) are each H. In another embodiment, R³ and R^(3′) are both H. Inanother embodiment, R⁴ and R^(4′) are both H. In another embodiment, oneof R³ and R^(3′) is C₁₋₃ alkyl while the other is H. In anotherembodiment, one of R⁴ and R^(4′) is C₁₋₃ alkyl while the other is H. Inanother embodiment, one of R³ and R^(3′) is C₁₋₃ alkyl and one of R⁴ andR^(4′) is C₁₋₃ alkyl while the others are H. In another embodiment, bothR³ and R^(3′) are independently C₁₋₃ alkyl. In another embodiment, bothR⁴ and R^(4′), are independently C₁₋₃ alkyl.

The alkylating activity of a compound of formula (I) or (II) or itscyclopropyl-containing analog may also be affected by the nature of X¹.The nature of X¹ may affect the rate at which and the conditions underwhich the seco agents ring close to the cyclopropyl analogs and/or therate at which the cyclopropyl ring is opened by nucleophilic attack (byDNA), and thus affect the alkylation behavior. In one embodiment, X¹ isO. In another embodiment, X¹ is NR¹³.

The substituents R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′), and X² as well asthe size of the ring(s) connected to the left-hand side of the ringbearing X¹ may for example, each independently or two or more takentogether, affect the pharmacokinetic properties of the agent, affect thewater solubility, affect the aggregation behavior, affect the DNAalkylation process, or affect the DNA binding strength.

Furthermore, especially R⁵ and R^(5′), and to some degree R⁶ and R^(6′)as well, may also affect the degree of shielding of the carbon on whichnucleophilic attack can occur.

In one embodiment, R⁵ and R^(5′) are both H, or R⁵ is H and R^(5′) isabsent. In another embodiment, at least one of R⁵ and R^(5′) is nothydrogen. In another embodiment, R⁵ is not hydrogen. In anotherembodiment, R⁵ is selected from OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN,C(O)NH₂, C(O)H, C(O)OH, halogen, R^(e), SR^(e), S(O)R^(e), S(O)₂R^(e),S(O)OR^(e), S(O)₂OR^(e), OS(O)R^(e), OS(O)₂R^(e), OS(O)OR^(e),OS(O)₂OR^(e), OR^(e), NHR^(e), N(R^(e))R^(f), ⁺N(R^(e))(R^(f))R^(g),P(O)(OR^(e))(OR^(f)), OP(O)(OR^(e))(OR^(f)), SiR^(e)R^(f)R^(g),C(O)R^(e), C(O)OR^(e), C(O)N(R^(e))R^(f), OC(O)R^(e), OC(O)OR^(e),OC(O)N(R^(e))R^(f), N(R^(e))C(O)R^(f), N(R^(e))C(O)OR^(f), andN(R^(e))C(O)N(R^(f))R^(g), wherein R^(e), R^(f), and R^(g) areindependently selected from H and optionally substituted C₁₋₃ alkyl,C₁₋₃ heteroalkyl, C₃ cycloalkyl, or C₁₋₃ heterocycloalkyl, two or moreof R^(e), R^(f), and R^(g) optionally being joined by one or more bondsto form one or more optionally substituted carbocycles and/orheterocycles. In another embodiment, R⁵ is selected from H, OH, SH, NH₂,N₃, NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH, halogen, R^(e), SR^(e),S(O)R^(e), S(O)₂R^(e), S(O)OR^(e), S(O)₂OR^(e), OS(O)R^(e), OS(O)₂R^(e),OS(O)OR^(e), OS(O)₂OR^(e), OR^(e), NHR^(e), N(R^(e))R^(f),⁺N(R^(e))(R^(f))R^(g), P(O)(OR^(e))(OR^(f)), OP(O)(OR^(e))(OR^(f)),SiR^(e)R^(f)R^(g), C(O)R^(e), C(O)OR^(e), C(O)N(R^(e))R^(f), OC(O)R^(e),OC(O)OR^(e), OC(O)N(R^(e))R^(f), N(R^(e))C(O)R^(f), N(R^(e))C(O)OR^(f),and N(R^(e))C(O)N(R^(f))R^(g), wherein R^(e), R^(f), and R^(g) areindependently selected from H and optionally substituted C₁₋₃ alkyl,C₁₋₃ heteroalkyl, C₃ cycloalkyl, or C₁₋₃ heterocycloalkyl, two or moreof R^(e), R^(f), and R^(g) optionally being joined by one or more bondsto form one or more optionally substituted carbocycles and/orheterocycles. In another embodiment, R⁵ is selected from H, OH, SH, NH₂,N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH, halogen, R^(e″), SR^(e),S(O)R^(e), S(O)₂R^(e), S(O)OR^(e), S(O)₂OR^(e), OS(O)R^(e), OS(O)₂R^(e),OS(O)OR^(e), OS(O)₂OR^(e), OR^(e), NHR^(e), N(R^(e))R^(f),⁺N(R^(e))(R^(f))R^(g), P(O)(OR^(e))(OR^(f)), OP(O)(OR^(e))(OR^(f)),SiR^(e)R^(f)R^(g), C(O)R^(e), C(O)OR^(e), C(O)N(R^(e))R^(f), OC(O)R^(e),OC(O)OR^(e), OC(O)N(R^(e))R^(f), N(R^(e))C(O)R^(f), N(R^(e′))C(O)OR^(f),and N(R^(e))C(O)N(R^(f))R^(g), wherein R^(e), R^(f), and R^(g) areindependently selected from H and optionally substituted C₁₋₃ alkyl,C₁₋₃ heteroalkyl, C₃ cycloalkyl, or C₁₋₃ heterocycloalkyl, R^(e″) isselected from H, substituted methyl, and optionally substituted C₂₋₃alkyl, C₁₋₃ heteroalkyl, C₃ cycloalkyl, or C₁₋₃ heterocycloalkyl, two ormore of R^(e), R^(f), and R^(g) optionally being joined by one or morebonds to form one or more optionally substituted carbocycles and/orheterocycles. In yet another embodiment, R⁵ is selected from nitro,halogen, amino, hydroxy, and optionally substituted C₁₋₃ alkylamino,di(C₁₋₃ alkyl)amino, C₁₋₃ alkylcarbonylamino, C₁₋₃ alkoxycarbonylamino,C₁₋₃ alkyloxy, C₁₋₃ alkylcarbonyloxy, C₁₋₃ alkylaminocarbonyloxy, orC₁₋₃ alkyl. In yet another embodiment, R⁵ is optionally substitutedlinear C₁₋₃ alkyl. In another embodiment, R⁵ is unsubstituted linearC₁₋₃ alkyl. In another embodiment, R⁵ is selected from methyl, ethyl,propyl, isopropyl, nitro, CF₃, F, Cl, Br, methoxy, amino (NH₂),methylamino, formyl, hydroxymethyl, and dimethylamino. In anotherembodiment, R⁵ is methyl. In other embodiments, R⁵ is ethyl or propyl orisopropyl.

In one embodiment, R⁶ and R^(6′) are hydrogen, or R⁶ is hydrogen andR^(6′) is absent. In another embodiment, at least one of R⁶ and R^(6′)is not hydrogen. In another embodiment, R⁶ is not hydrogen.

In another embodiment, R⁶ is a sterically bulky group, meaning that theatom of R⁶ that is connected to the remainder of a compound of formula(I) or (II) contains at least two substituents other than hydrogen orfluorine that are each connected via a single bond to said atom. In yetanother embodiment, R⁶ is selected from tert-butyl and isopropyl. Inanother embodiment, R⁶ is tert-butyl. In yet another embodiment, R⁶ isisopropyl.

Although the alkylation rate and efficiency of compounds of formulae (I)and (II) may be tuned in several ways, in one aspect of this invention,this is achieved by introducing steric shielding choosing for a compoundof formula (I) at least one of R², R³, R^(3′), R⁴, R^(4′), R⁵, R^(5′),R⁶, and R^(6′) present to be other than hydrogen and for a compound offormula (II) optionally one or more of R², R³, R¹, R⁴, R^(4′), R⁵,R^(5′), R⁶, and R^(6′) present to be other than hydrogen. Substituentsshould not cause too much steric hindrance, however, especially whenmore than one of these substituents is other than hydrogen, as thismight adversely affect DNA alkylation. Furthermore, it may provide forless efficient binding in the DNA minor groove and may pose syntheticdifficulties. In one embodiment, only one of R², R³, R^(3′), R⁴, R^(4′),R⁵, R^(5′), R⁶, and R^(6′) present is other than hydrogen. In anotherembodiment, two of R², R³, R^(3′), R⁴, R^(4′), R⁵, R^(5′), R⁶, andR^(6′) present are other than hydrogen and at least one of these two isa methyl group or a group of similar size, meaning that its van derWaals radius is no more than twice as large as that of a methyl group.In yet another embodiment, three of R², R³, R^(3′), R⁴, R^(4′), R⁵,R^(5′), R⁶, and R^(6′) present are other than hydrogen and these threeeach independently are a methyl group or a group of similar size,meaning that its van der Waals radius is no more than twice as large asthat of a methyl group.

In one aspect of this invention, compounds of formulae (I) and (II) arerepresented by compounds of formulae (Ia) and (IIa), respectively:

In one embodiment, R⁶ and R⁷ in (Ia) or (IIa) are both H.

R⁶ may be a sterically bulky group such that the carbon bearing R¹ issomewhat shielded. In one embodiment, R⁶ is a tert-butyl group; inanother embodiment, R⁶ is an isopropyl group.

In another aspect, compounds of formulae (I) and (II) are represented bycompounds of formulae (Ib) and (IIb), respectively:

In one embodiment, X² in (Ib) or (IIb) is N.

In another embodiment, X² in (Ib) or (IIb) is CH.

In another embodiment, R⁵, R⁶, and R⁷ in (Ib) or (IIb) are each H.

In another embodiment, R⁵, R⁶, and R⁷ in (Ib) or (IIb) are each H and X²is CH.

In another embodiment, R⁵ and R⁷ in (Ib) or (IIb) are each H and R⁶ isCO₂Me.

In another embodiment, R⁵ and R⁷ in (Ib) or (IIb) are each H and R⁶ isOMe.

In another embodiment, R⁵ and R⁷ in (Ib) or (IIb) are each H and R⁶ isCN.

In yet another embodiment, R⁵ in (Ib) or (IIb) is selected from nitro,halogen, amino, hydroxy, and optionally substituted C₁₋₃ alkylamino,di(C₁₋₃ alkyl)amino, C₁₋₃ alkylcarbonylamino, C₁₋₃ alkoxycarbonylamino,C₁₋₃ alkyloxy, C₁₋₃ alkylcarbonyloxy, C₁₋₃ alkylaminocarbonyloxy, orC₁₋₃ alkyl. In yet another embodiment, R⁵ in (Ib) or (IIb) is optionallysubstituted linear C₁₋₃ alkyl. In another embodiment, R⁵ in (Ib) or(IIb) is unsubstituted linear C₁₋₃ alkyl. In another embodiment, R⁵ in(Ib) or (IIb) is methyl. In other embodiments, R⁵ in (Ib) or (IIb) isethyl or propyl or isopropyl.

In yet another aspect, compounds of formulae (I) and (II) arerepresented by compounds of formulae (Ic) and (IIc), respectively:

In one embodiment, X² in (Ic) or (IIc) is NH.

In another embodiment, R⁶ and R⁷ in (Ic) or (IIc) are H and CO₂CH₃,respectively, and X² is NH.

In another embodiment, R⁷ and R⁶ in (Ic) or (IIc) are H and CO₂CH₃,respectively, and X² is NH.

In another embodiment, R⁶ in (Ic) or (IIc) is CH₃ and X² is NH.

In yet another aspect, compounds of formulae (I) and (II) arerepresented by compounds of formulae (Id) and (IId), respectively:

In one embodiment, X² in (Id) or (IId) is NH.

In another embodiment, R⁶ and R^(6′) in (Id) or (IId) together are ═O.

In another embodiment, R⁷ and R^(7′) in (Id) or (IId) are CO₂CH₃ andCH₃, respectively.

In another embodiment, in a compound of formula (Id) or (IId), X² is NH,R⁶ and R^(6′) together are ═O, and R⁷ and R^(7′) are CO₂CH₃ and CH₃,respectively.

In one embodiment, c=1 and b=1. In another embodiment, c=1 and b=0. Inanother embodiment, a=0.

In one embodiment, R² in a compound of formula (I) is optionallysubstituted C₁₋₃ alkyl and R³, R^(3′), R⁴, R^(4′), R⁵, R^(5′), R⁶, andR^(6′) present are hydrogen. In another embodiment, R² in a compound offormula (I) is selected from methyl, ethyl, propyl, and isopropyl, andR³, R^(3′), R⁴, R^(4′), R⁵, R^(5′), R⁶, and R^(6′) present are eachhydrogen. In a further embodiment, R² in a compound of formula (I) isselected from N₃, halogen, OS(O)₂R^(a), CF₃, NO₂, N(R^(b))R^(c), OR^(b),C(O)R^(a), C(O)OR^(a), OC(O)R^(a), OC(O)OR^(a), OC(O)N(R^(a))R^(a′), andC(O)N(R^(a))R^(a′) and R³, R^(3′), R⁴, R^(4′), R⁵, R^(5′), R⁶, andR^(6′) present are each hydrogen. In another embodiment, R² in acompound of formula (I) is selected from chloride and bromide and R³,R^(3′), R⁴, R^(4′), R⁵, R^(5′), R⁶. and R^(6′) present are eachhydrogen.

In one embodiment, R², R⁵, R^(5′), R⁶, and R^(6′) present are eachhydrogen and one of R³, R^(3′), R⁴, and R^(4′) is selected from C₁₋₃alkyl. In another embodiment, R², R⁵, R^(5′), R⁶, and R^(6′) present areeach hydrogen and one of R³, R^(3′), R⁴, and R^(4′) is selected frommethyl. In another embodiment, R², R⁵, R^(5′), R⁶, and R^(6′) presentare each hydrogen and two of R³, R^(3′), R⁴, and R^(4′) areindependently selected from C₁₋₃ alkyl. In yet another embodiment, R²,R⁵, R^(5′), R⁶ and R^(6′) present are each hydrogen and two of R³,R^(3′), R⁴, and R^(4′) are methyl.

In yet another embodiment, R⁵ is selected from nitro, halogen, amino,hydroxy, and optionally substituted C₁₋₃ alkylamino, di(C₁₋₃alkyl)amino, C₁₋₃ alkylcarbonylamino, C₁₋₃ alkoxycarbonylamino, C₁₋₃alkyloxy, C₁₋₃ alkylcarbonyloxy, C₁₋₃ alkylaminocarbonyloxy, or C₁₋₃alkyl, and R², R³, R^(3′), R⁴, R^(4′), R^(5′), R⁶ . . . and R^(6′)present are each H. In yet another embodiment, R⁵ is optionallysubstituted linear C₁₋₃ alkyl and R², R³, R^(3′), R⁴, R^(4′), R^(5′),R⁶, and R^(6′) present are each hydrogen. In another embodiment, R⁵ isunsubstituted linear C₁₋₃ alkyl and R², R³, R^(3′), R⁴, R^(4′), R^(5′),R⁶, and R^(6′) present are each hydrogen. In another embodiment, R⁵ isselected from methyl, ethyl, propyl, isopropyl, nitro, CF₃, F, Cl, Br,methoxy, amino, methylamino, formyl, hydroxymethyl, and dimethylamino,and R², R³, R^(3′), R⁴, R^(4′), R^(5′), R⁶, and R^(6′) present are eachhydrogen. In another embodiment, R⁵ is not methyl when R⁸ isN,N-dimethylaminoethoxy, R⁹, R¹⁰, and R¹¹ are hydrogen, X³ is NH, X⁴ isCH, and X⁵ is O.

In another embodiment, R⁶ is a sterically bulky group and R², R³,R^(3′), R⁴, R^(4′), R⁵, R^(5′), and R^(6′) present are each hydrogen. Inone embodiment, R⁶ is tert-butyl and R², R³, R^(3′), R⁴, R^(4′), R⁵,R^(5′), and R^(6′) present are each hydrogen. In another embodiment, R⁶is isopropyl and R², R³, R^(3′), R⁴, R^(4′), R⁵R^(5′), and R^(6′)present are each hydrogen. In yet another embodiment, R⁶ is selectedfrom tert-butyl and isopropyl, and b=0. In yet another embodiment, R⁶ isselected from tert-butyl and isopropyl, and c=0.

Steric hindrance may be introduced by choosing R⁵ to be other thanhydrogen. In one embodiment, a compound of formula (I) is represented bya compound of formula (Ie):

wherein DA is

or by an isomer, or by a mixture of isomers, wherein R^(1a) is selectedfrom Cl and Br and R^(5a) is selected from nitro, halogen, amino,hydroxy, and optionally substituted C₁₋₃ alkylamino, di(C₁₋₃alkyl)amino, C₁₋₆ alkylcarbonylamino, C₁₋₃ alkoxycarbonylamino, C₁₋₃alkyloxy, C₁₋₃ alkylcarbonyloxy, C₁₋₃ alkylaminocarbonyloxy, or C₁₋₃alkyl.

In a further embodiment, a compound of formula (I) is represented by acompound of formula (Ie) wherein DA is

or by an isomer, or by a mixture of isomers, wherein R^(1a) is selectedfrom Cl and Br and R^(5a′) is selected from methyl, ethyl, propyl,isopropyl, nitro, CF₃, F, Cl, Br, methoxy, amino, methylamino, formyl,hydroxymethyl, and dimethylamino.

In further embodiments, a compound of formula (I) is represented by acompound of formula (Ie) wherein DA is

or by an isomer of one of these, or by a mixture of isomers.

Alternatively, R² may be selected to be other than hydrogen in order tointroduce steric hindrance. In one embodiment, a compound of formula (I)is represented by a compound of formula (Ie) wherein DA is

or by an isomer, or by a mixture of isomers, wherein R^(1a) is selectedfrom Cl and Br and R^(2a) is selected from Cl and Br.

In further embodiments, a compound of formula (I) is represented by acompound of formula (Ie) wherein DA is

or by an isomer of one of these, or by a mixture of isomers.

Alternatively again, steric hindrance may also be created by choosing R⁶to be a relatively bulky substituent. In one embodiment, a compound offormula (I) is represented by a compound of formula (Ie) wherein DA is

or by an isomer of one of these, or by a mixture of isomers.

More than one of R², R³, R^(3′), R⁴, R^(4′), R⁵, R^(5′), R⁶, and R^(6′)present may be other than hydrogen. For example, R² and R⁵ may beselected to be other than hydrogen. Alternatively, R² and R⁶, or R⁶ andR⁵, or R² and R⁴, or R² and R^(4′) may for example be selected to beother than hydrogen. All other combinations of two or more substituentsfrom the group of R², R³, R^(3′), R⁴, R^(4′), R⁵, R^(5′), R⁶, and R^(6′)may be selected to be other than hydrogen as well.

In distinct embodiments, a compound of formula (I) is represented by acompound of formula (Ie) wherein DA is

or by an isomer of one of these, or by a mixture of isomers.

In another aspect, this invention relates to a DNA alkylating compoundthat comprises the moiety

wherein R¹, R², R³, R^(3′), R⁴, R^(4′), R^(5′), R⁶, R^(6′), R⁷, R^(7′),X¹, X², a, b, and c are as defined for a compound of formula (I) or (II)hereinabove, and wherein R⁵ is selected from OH, SH, NH₂, N₃, NO₂, NO,CF₃, CN, C(O)NH₂, C(O)H, C(O)OH, halogen, R^(e), SR^(e), S(O)R^(e),S(O)₂R^(e), S(O)OR^(e), S(O)₂OR^(e), OS(O)R^(e), OS(O)₂R^(e),OS(O)OR^(e), OS(O)₂OR^(e), OR^(e), NHR^(e), N(R^(e))R^(f),⁺N(R^(e))(R^(f))R^(g), P(O)(OR^(e))(OR^(f)), OP(O)(OR^(e))(OR^(f)),SiR^(e)R^(f)R^(g), C(O)R^(e), C(O)OR^(e), C(O)N(R^(e))R^(f), OC(O)R^(e),OC(O)OR^(e), OC(O)N(R^(e))R^(f), N(R^(e))C(O)R^(f), N(R^(e))C(O)OR^(f),and N(R^(e))C(O)N(R^(f))R^(g), wherein R^(e), R^(f), and R^(g) areindependently selected from H and optionally substituted C₁₋₃ alkyl,C₁₋₃ heteroalkyl, C₃ cycloalkyl, or C₁₋₃ heterocycloalkyl, two or moreof R^(e), R^(f), and R^(g) optionally being joined by one or more bondsto form one or more optionally substituted carbocycles and/orheterocycles.

The DNA-binding unit of compounds of formulae (I) and (II) is consideredto assist in efficient binding of these compounds to DNA. It comprisesone (hetero)aromatic structure or more (hetero)aromatic structures fusedor coupled together. The heteroaromatic moiety can for example be anoptionally substituted indole or benzofuran moiety. Such a moiety may becoupled to a DNA-alkylating moiety via, for instance, an amide bond. Inone embodiment, X³ is NH. In another embodiment, X³ is O. In anotherembodiment, X⁴ is CH. In another embodiment, X⁵ is O. In anotherembodiment, X³ is NH, X⁴ is CH, and X⁵ is O.

Substituents R⁸, R⁹, R¹⁰, and R¹¹ may help improve the binding affinityof a compound of formula (I) or (II) for DNA. This may for example occurby selecting one of these substituents to be a (hetero)aromatic moiety.Thus, in one embodiment, R⁸ is selected from

wherein X^(3″) is selected from O, S, and NR^(15′), wherein R^(15′) isselected from the same pool of substituents as R¹⁵, R^(q) and R^(r) areselected from H and optionally substituted C₁₋₃ alkyl, and NR^(q)R^(r)is connected to the phenyl ring through any one of carbon atoms 1-4. Inanother embodiment, NR^(q)R^(r) is connected to the phenyl ring throughcarbon atom 2 or 4.

In another embodiment, R⁸ is a substituent of the formula:

wherein X^(3′), X^(4′), X^(5′), R^(8′), R^(9′), R^(10′), and R^(11′) areselected from the same pool of substituents as defined above for X³, X⁴,X⁵, R⁸, R⁹, R¹⁰, and R¹¹, respectively, and wherein R^(8″) is selectedfrom H and optionally substituted C₁₋₅ alkyl or C₁₋₅ heteroalkyl andoptionally joined with R⁹ or R¹¹ to form an optionally substitutedheterocycle.

All embodiments for R⁸, R⁹, R¹⁰, and R¹¹ specified in this document arealso applicable to R^(8′), R^(9′), R^(10′), and R^(11′), respectively.

In one embodiment, R⁸ or R^(8″) and R¹¹, and/or R^(8′) and R^(11′) arejoined to form together with the linking atoms an optionally substituteddihydropyrrole moiety.

Substituents R⁸, R⁹, R¹⁰, and R¹¹ may also assist in improving thepharmacokinetic properties of a compound of formula (I) or (II) or itsconjugate, for example, its water solubility. This may for example occurby selecting one or more of the substituents R⁸, R⁹, R¹⁰, and R¹¹ tocomprise or be a water-soluble group. Furthermore, such a water-solublegroup may prevent a compound of formula (I) or (II) from crossing abiological barrier, especially when it is an apolar barrier, such as acell membrane. This may be advantageous, especially when a compound offormula (I) or (II) is delivered into a targeted cell throughconjugation to a targeting moiety before it is released from theconjugate. When a compound of formula (I) or (II) is prematurelyreleased from the conjugate, e.g., in the circulation, it may be unableor only moderately able to enter (non-targeted) cells aspecifically asits membrane translocation capabilities may be impaired. This may leadto increased selectivity and therefore to fewer side effects. Inaddition, at least in some instances, for example when the water-solublegroup is positively charged under physiological conditions, thewater-soluble group may also improve the binding affinity for DNA bymeans of favorable electrostatic interactions.

A water-soluble group is a group that imparts increased solubility on acompound of formula (I) or (II) and/or a conjugate thereof. In oneembodiment, water solubility of a compound of this invention carrying awater-soluble group is increased by more than 100% compared to thecompound lacking said water-soluble group. In other embodiments, watersolubility of a compound of this invention carrying a water-solublegroup is increased by more than 75% or 50% or 25% or 10% compared to thecompound lacking said water-soluble group. The water-soluble group mayalso contribute to prevent or reduce aggregation of compounds of thisinvention or to reduce side effects. Examples of water-soluble groupsinclude, but are not limited to, —NH₂, —NH—, —NHR^(s), —NR^(s)—,—N(R^(s))(R^(t)), —⁺N(R^(s))(R^(t))—, —⁺N(R^(s))(R^(t))(R^(u)), —COOH,—OP(O)(OH)₂, —OP(O)(OH)O—, —OP(O)(OR^(s))O—, —OP(O)(OH)OR^(s)),—OP(O)(OR^(s))OR^(t), —P(O)(OH)₂, —P(O)(OH)O—, —P(O)(OR^(s))OH,—P(O)(OR^(s))O—, —P(O)(OR^(s))(OR^(t)), —OS(O)₂OH, —OS(O)₂O—,—OS(O)₂OR^(s), —S(O)₂OH, —S(O)₂O—, —S(O)₂OR^(s), —OS(O)OH, —OS(O)O—,—OS(O)OR^(s), —S(O)OH, —S(O)O—, —OS(O)—, —S(O)OR^(s), —OS(O)₂—,—OS(O)₂R^(s), —S(O)₂—, —S(O)₂R^(s), —OS(O)R^(s), —S(O)—, —S(O)R^(s),—(OCH₂CH₂)_(v′)OH, —(OCH₂CH₂)_(v′)O—, —(OCH₂CH₂)_(v′)OR^(s), a sugarmoiety, an oligosaccharide moiety, and an oligopeptide moiety, or aprotonated or deprotonated form thereof and further any combinationthereof, wherein R^(s), R^(t), and R^(u) are independently selected fromH and optionally substituted C₁₋₃ alkyl, two or more of R^(s), R^(t),and R^(u) optionally being joined by one or more bonds to form one ormore carbocycles and/or heterocycles, and v′ is an integer selected from1 to 100. The water-soluble group may be at any position within R⁸, R⁹,R¹⁰, and/or R¹¹ or may constitute the whole R⁸, R⁹, R¹⁰, or R¹¹ moiety.The water-soluble group may for example be located at any interiorposition, be part of the main chain, be part of a ring structure, be afunctional group pending to the main chain or a ring, or be placed atthe position at which the R⁸, R⁹, R¹⁰, or R¹¹ substituent is attached tothe remainder of the agent.

In one embodiment, none of R⁸, R⁹, R¹⁰, and R¹¹ contains a water-solublegroup.

In another embodiment, at least one of R⁸, R⁹, R¹⁰, and R¹¹ contains awater-soluble group.

In another embodiment, at least one of R⁸, R⁹, and R¹⁰ contains awater-soluble group.

In yet another embodiment, R⁸ contains a water-soluble group.

In yet another embodiment, R⁹ contains a water-soluble group.

In yet another embodiment, R¹⁰ contains a water-soluble group.

In one embodiment, the water-soluble group is a carboxylic acid group.

In another embodiment, the water-soluble group is an amino group.

In a further embodiment, the water-soluble group is a primary aminogroup.

In another embodiment, the water-soluble group is a secondary aminogroup.

In another embodiment, the water-soluble group is a tertiary aminogroup.

In another embodiment, the water-soluble group is a quatemary amino(ammonium) group.

In other embodiments, the water-soluble group is a primary or secondaryor tertiary or quaternary aliphatic amino group.

In other embodiments, the water-soluble group is a dimethylamino groupor a methylamino group or an amino (NH₂) group.

In another embodiment, the water-soluble group is anN-methyl-N-(carboxymethyl)amino group.

In yet another embodiment, the water-soluble group is anN-methyl-N-(2-methoxy-2-oxoethyl)amino group.

In another embodiment, at least one of the substituents R¹, R², R³,R^(3′), R⁴, R^(4′), R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′), R⁸, R⁹, R¹⁰,R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ contains or is the moietyCOOH.

In another embodiment, at least one of the substituents R⁸, R⁹, and R¹⁰contains or is the moiety COOH and there is at least anotherwater-soluble group present in R⁸, R⁹, or R¹⁰.

In another embodiment, at least one of the water-soluble groups in R⁸,R⁹, and R¹⁰ is an aliphatic secondary, tertiary, or quaternary aminemoiety not being conjugated to an aromatic moiety or a carbonyl group.

In yet another embodiment, at least one of the water-soluble groups inR⁸, R⁹, R¹⁰, and R¹¹ is an aliphatic secondary amine moiety not beingconjugated to an aromatic moiety or a carbonyl group and at least one ofthe substituents R¹, R², R³, R^(3′), R⁴, R^(4′), R⁵, R^(5′), R⁶, R^(6′),R⁷, R^(7′), R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸contains or is the moiety COOH.

In yet another embodiment, at least one of the water-soluble groups inR⁸, R⁹, and R¹⁰ is an aliphatic secondary amine moiety not beingconjugated to an aromatic moiety or a carbonyl group and at least one ofthe substituents R⁸, R⁹, and R¹⁰ contains or is the moiety COOH.

In yet another embodiment at least one of the substituents R⁸, R⁹, andR¹⁰ contains a COOH moiety and an aliphatic secondary amine moiety notbeing conjugated to an aromatic moiety or a carbonyl group.

In one embodiment, R⁸, R⁹, or R¹⁰ is selected from —O—C₁₋₆alkylene-N(R¹⁰⁰)₂, —N(R¹⁰⁰)C(O)—C₁₋₅ alkylene-N(R¹⁰⁰)₂,(1-(R¹⁰⁰)piperidin-4-yl)-C₁₋₅ is alkylene-O—, (morpholin-4-yl)-C₁₋₈alkylene-O—, —C(O)N(R¹⁰⁰)—C₁₋₆ alkylene-N(R¹⁰⁰)₂, and —CO₂R¹⁰⁰, whereineach R¹⁰⁰ is independently selected from H and C₁₋₃ alkyl, the latterbeing optionally substituted with COOH or COOR³⁰⁰, R³⁰⁰ being C₁₋₄alkyl.

In another embodiment, R⁸ is selected from —O—C₁₋₆ alkylene-N(R¹⁰⁰)₂,—N(R¹⁰⁰)C(O)—C₁₋₅ alkylene-N(R¹⁰⁰)₂, (1-(R¹⁰⁰)piperidin-4-yl)-C₁₋₅alkylene-O—, (morpholin-4-yl)-C₁₋₈ alkylene-O—, —C(O)N(R¹⁰⁰)—C₁₋₆alkylene-N(R¹⁰⁰)₂, and —CO₂R¹⁰⁰, wherein each R¹⁰⁰ is independentlyselected from H and C₁₋₃ alkyl, the latter being optionally substitutedwith COOH or COOR³⁰⁰, R³⁰⁰ being C₁₋₄ alkyl.

In another embodiment, R⁹ is selected from —O—C₁₋₆ alkylene-N(R¹⁰⁰)₂,—N(R¹⁰⁰)C(O)—C₁₋₅ alkylene-N(R¹⁰⁰)₂, (1-(R¹⁰⁰)piperidin-4-yl)-C₁₋₅alkylene-O—, (morpholin-4-yl)-C₁₋₈ alkylene-O—, —C(O)N(R¹⁰⁰)—C₁₋₆alkylene-N(R¹⁰⁰)₂, and —CO₂R¹⁰⁰, wherein each R¹⁰⁰ is independentlyselected from H and C₁₋₃ alkyl, the latter being optionally substitutedwith COOH or COOR³⁰⁰, R³⁰⁰ being C₁₋₄ alkyl.

In another embodiment, R¹⁰ is selected from —O—C₁₋₆ alkylene-N(R¹⁰⁰)₂,—N(R¹⁰⁰)C(O)—C₁₋₅ alkylene-N(R¹⁰⁰)₂, (1-(R¹⁰⁰)piperidin-4-yl)-C₁₋₅alkylene-O—, (morpholin-4-yl)-C₁₋₈ alkylene-O—, —C(O)N(R¹⁰⁰)—C₁₋₆alkylene-N(R¹⁰⁰)₂, and —CO₂R¹⁰⁰, wherein each R¹⁰⁰ is independentlyselected from H and C₁₋₃ alkyl, the latter being optionally substitutedwith COOH or COOR³⁰⁰, R³⁰⁰ being C₁₋₄ alkyl.

In one embodiment, R⁸, R⁹, or R¹⁰ is selected from

In another embodiment, R⁸ is selected from

In yet another embodiment, R⁹ is selected from

In another embodiment, R⁸ is selected from

Either none or one or more of R⁸, R⁹, R¹⁰, and R¹¹ may be selected to beother than hydrogen. For example, one of R⁸, R⁹, R¹⁰, and R¹¹ may beother than hydrogen. In one embodiment, R⁸ is other than hydrogen andR⁹, R¹⁰, and R¹¹ are hydrogen. In another embodiment, R⁹ is other thanhydrogen and R⁸, R¹⁰, and R¹¹ are hydrogen. Alternatively, two of R⁸,R⁹, R¹⁰, and R¹¹ may be other than hydrogen. In one embodiment, R⁸ andR⁹ are other than hydrogen and R¹⁰ and R¹¹ are hydrogen. In anotherembodiment, R⁸ and R¹⁰ are other than hydrogen and R⁹ and R¹¹ arehydrogen. As another alternative, three of R⁸, R⁹, R¹⁰, and R¹¹ areother than hydrogen. In one embodiment, R⁸, R⁹, and R¹⁰ are other thanhydrogen and R¹ is hydrogen.

In one embodiment of this invention, a compound of formula (I) or (II)is represented by a compound of formula (If) or (IIf), respectively:

wherein DB is

wherein X³, X⁴, and X⁵ are as defined hereinabove, R^(8a) is selectedfrom —O—C alkylene-N(R¹⁰⁰)₂, —N(R¹⁰⁰)C(O)—C₁₋₅ alkylene-N(R¹⁰⁰)₂,(1-(R¹⁰⁰)piperidin-4-yl)-C₁₋₅ alkylene-O—, (morpholin-4-yl)—C₁₋₈alkylene-O—, —C(O)N(R¹⁰⁰)—C₁₋₆ alkylene-N(R¹⁰⁰)₂, and —CO₂R¹⁰⁰, whereineach R¹⁰⁰ is independently selected from H and C₁₋₃ alkyl, the latterbeing optionally substituted with COOH or COOR³⁰⁰, R³⁰⁰ being C₁₋₄alkyl, and R^(9a) is selected from H, C₁₋₃ alkyloxy, C₁₋₃ alkylcarbonyl.In another embodiment of this invention, a compound of formula (I) and(II) is represented by a compound of formula (If) and (IIf),respectively, wherein DB is

wherein X³, X⁴, and X⁵ are as defined hereinabove, R^(9b) is selectedfrom —O—C₁₋₆ alkylene-N(R¹⁰⁰)₂, —N(R¹⁰⁰)C(O)—C₁₋₅ alkylene-N(R¹⁰⁰)₂,(1-(R¹⁰⁰)piperidin-4-yl)-C₁₋₅ alkylene-O—, (morpholin-4-yl)-C₁₋₈alkylene-O—, —C(O)N(R¹⁰⁰)—C₁₋₆ alkylene-N(R¹⁰⁰)₂, and —CO₂R¹⁰⁰, whereineach R¹⁰⁰ is independently selected from H and C₁₋₃ alkyl, the latterbeing optionally substituted with COOH or COOR³⁰⁰, R³⁰⁰ being C₁₋₄alkyl, and R^(8b) is selected from H, C₁₋₃ alkyloxy, and C₁₋₃alkylcarbonyl.

In another embodiment of this invention, a compound of formula (I) or(II) is represented by a compound of formula (If) or (IIf),respectively, wherein DB is

wherein X³, X⁴, and X⁵ are as defined hereinabove, R^(8c) is selectedfrom —O—C₁₋₆ alkylene-N(R¹⁰⁰)₂, —N(R¹⁰⁰)C(O)—C₁₋₅ alkylene-N(R¹⁰⁰)₂,(1-(R¹⁰⁰)piperidin-4-yl)-C₁₋₅ alkylene-O—, (morpholin-4-yl)-C₁₋₈alkylene-O—, —C(O)N(R¹⁰⁰)—C₁₋₆ alkylene-N(R¹⁰⁰)₂, and —CO₂R¹⁰⁰, whereineach R¹⁰⁰ is independently selected from H and C₁₋₃ alkyl, the latterbeing optionally substituted with COOH or COOR³⁰⁰, R³⁰⁰ being C₁₋₄alkyl, and R^(10c) is selected from H, C₁₋₃ alkyloxy, and C₁₋₃alkylcarbonyl.

In another embodiment of this invention, a compound of formula (I) or(II) is represented by a compound of formula (If) or (IIf),respectively, wherein DB is

wherein X³, X⁴, and X⁵ are as defined hereinabove, R^(10d) is selectedfrom —O—C₁ . . . alkylene-N(R¹⁰⁰)₂, —N(R¹⁰⁰)C(O)—C₁₋₅ alkylene-N(R¹⁰⁰)₂,(1-(R^(φ))piperidin-4-yl)-C₁₋₅ alkylene-O—, (morpholin-4-yl)-C₁₋₈alkylene-O—, —C(O)N(R¹⁰⁰)—C₁₋₆, alkylene-N(R¹⁰⁰)₂, and —CO₂R¹⁰⁰, whereineach R¹⁰⁰ is independently selected from H and C₁₋₃ alkyl, the latterbeing optionally substituted with COOH or COOR³⁰⁰, R³⁰⁰ being C₁₋₄alkyl, and R^(8d) is selected from H, C₁₋₃ alkyloxy, and C₁₋₃alkylcarbonyl.

In distinct embodiments, a compound of formula (I) or (II) isrepresented by a compound of formula (If) or (IIf), respectively,wherein DB is

In other distinct embodiments, a compound of formula (I) or (II) isrepresented by a compound of formula (If) or (IIf), respectively,wherein DB is

In other distinct embodiments, a compound of formula (I) or (II) isrepresented by a compound of formula (If) or (IIf), respectively,wherein DB is

In distinct embodiments, a compound of formula (I) is represented by

or by an isomer of one of these, or by a mixture of isomers, whereinR^(5b) is selected from the same pool as R^(5a′), except that it may beselected to be hydrogen as well, R^(6b) is selected from hydrogen,tert-butyl, and isopropyl, R^(2b) is selected from the same pool as R²,except that it may not be methyl when R^(5b) and R^(6b) are bothhydrogen, R^(1a), R^(8b), R^(8d), R^(9a), R^(9b), R^(10c), and R^(10d)are as defined above, and R^(8a′) and R^(8c′) are selected from the samepools of substituents as R^(8a) and R^(8c), respectively, except thatR^(8a′) and R^(8c′) may not be N,N-dimethylaminoethoxy when R^(9a) orR^(10c) is H, R^(5b) is methyl, R^(1a) is chloride, and R^(2b) andR^(6b) are both hydrogen.

In distinct embodiments, a compound of formula (I) is represented by

or by an isomer of one of these, or by a mixture of isomers, whereinR^(1a), R^(5a′), R^(8b), R^(8d), R^(9a), R^(9b), R^(10c), and R^(10d)are as defined above and R^(8a′) is selected from the same pool ofsubstituents as R^(8a) except that R^(8a′) and R^(8c′) may not beN,N-dimethylaminoethoxy when R^(9a) or R^(10c) is H, R^(1a) is chloride,and R^(5a′) is methyl.

In other distinct embodiments, a compound of formula (I) is representedby

or by an isomer of one of these, or by a mixture of isomers, whereinR^(1a), R^(2a), R^(8a), R^(8b), R^(8c), R^(8d), R^(9a), R^(9b), R^(10c),and R^(10d) are as defined above.

In other distinct embodiments, a compound of formula (I) is representedby

or by an isomer of one of these, or by a mixture of isomers, whereinR^(1a), R^(8a), R^(8b), R^(8c), R^(8d), R^(9a), R^(9b), R^(10c), andR^(10d) are as defined above and R^(6a) is selected from tert-butyl andisopropyl.

In one embodiment, this invention relates to a compound of formula (I)or (II) wherein R² is selected from CF₃, CN, C(O)NH₂, C(O)H, C(O)OH,C(O)R^(a), C(O)OR^(a), and C(O)N(R^(a))R^(a′), wherein R^(a) and R^(a′)are independently selected from H and optionally substituted C₁₋₃ alkylor C₁₋₃ heteroalkyl, R³, R^(3′), R⁴, and R^(4′) are independentlyselected from hydrogen and optionally substituted C₁₋₃ alkyl, R¹ isselected from halogen and OSO₂R^(o), and at least one of R⁸, R⁹, R¹⁰,and R¹¹ comprises at least one water-soluble group.

In one embodiment, this invention relates to a compound of formula (Ib)or (IIb) wherein R² is selected from CF₃, CN, C(O)NH₂, C(O)H, C(O)OH,C(O)R^(a), C(O)OR^(a), and C(O)N(R^(a))R^(a′), wherein R^(a) and R^(a′)are independently selected from H and optionally substituted C₁₋₃ alkylor C₁₋₃ heteroalkyl; X² is CR¹⁴; R³, R^(3′), R⁴, and R^(4′) areindependently selected from hydrogen and optionally substituted C₁₋₃alkyl; R¹ is selected from halogen and OSO₂R^(o), provided that at leastone of R⁸, R⁹, R¹⁰, and R¹¹ comprises at least one water-soluble group.

In another embodiment, this invention relates to a compound of formula(I) or (II) wherein R² is selected from N₃, NO₂, NO, halogen, SR^(b),S(O)R^(a), S(O)₂R^(a), S(O)OR^(a), S(O)₂OR^(a), OS(O)R^(a), OS(O)₂R^(a),OS(O)OR^(a), OS(O)₂OR^(a), OR^(b), N(R^(b))R^(c), ⁺N(R^(b))(R^(c))R^(d),P(O)(OR^(a))(OR^(a′)), OP(O)(OR^(a))(OR^(a′)), SiR^(a)R^(a′)R^(a″),OC(O)R^(a), OC(O)OR^(a), OC(O)N(R^(a))R^(a′), N(R^(a))C(O)R^(a),N(R^(a))C(O)OR^(a′), and N(R^(a))C(O)N(R^(a′))R^(a′), wherein R^(a),R^(a′), and R^(a″) are independently selected from H and optionallysubstituted C₁₋₃ alkyl or C₁₋₃ heteroalkyl, and R^(b), R^(c), and R^(d)are independently selected from optionally substituted C₁₋₃ alkyl andC₁₋₃ heteroalkyl.

In another embodiment, this invention relates to a compound of formula(Ib) or (IIb) wherein R² is selected from N₃, NO₂, NO, halogen, SR^(b),S(O)R^(a), S(O)₂R^(a), S(O)OR^(a), S(O)₂OR^(a), OS(O)R^(a), OS(O)₂R^(a),OS(O)OR^(a), OS(O)₂OR^(a), OR^(b), N(R^(b))R^(c), ⁺N(R^(b))(R^(c))R^(d),P(O)(OR^(a))(OR^(a′)), OP(O)(OR^(a))(OR^(a′)), SiR^(a)R^(a′)R^(a″),OC(O)R^(a), OC(O)OR^(a), OC(O)N(R^(a))R^(a), N(R^(a))C(O)R^(a′),N(R^(a))C(O)OR^(a′), and N(R^(a))C(O)N(R^(a′))R^(a″), wherein R^(a),R^(a′), and R^(a″) are independently selected from H and optionallysubstituted C₁₋₃ alkyl or C₁₋₃ heteroalkyl, and R^(b), R^(c), and R^(d)are independently selected from optionally substituted C₁₋₃ alkyl andC₁₋₃ heteroalkyl, and X² is CR¹⁴.

In another embodiment, this invention relates to a compound of formula(I) or (II) wherein R², R³, R^(3′), R⁴, and R^(4′) are H, R⁵ and R^(5′)are independently selected from OH, SH, NH₂, N₃, NO₂, NO, halogen,SR^(e), S(O)R^(e), S(O)₂R^(e), S(O)OR^(e), S(O)₂OR^(e), OS(O)R^(e),OS(O)₂R^(e), OS(O)OR^(e), OS(O)₂OR^(e), OR^(e), NHR^(e), N(R^(e))R^(f),⁺N(R^(e))(R^(f))R^(g), P(O)(OR^(e))(OR^(f)), OP(O)(OR^(e))(OR^(f)),SiR^(e)R^(f)R^(g), OC(O)R^(e), N(R^(e))C(O)R^(f), N(R^(e))C(O)OR^(f),and N(R^(e))C(O)N(R^(f))R^(g), wherein R^(e), R^(f), and R^(g) areindependently selected from H and optionally substituted C₁₋₃ alkyl,C₁₋₃ heteroalkyl, C₃ cycloalkyl, or C₁₋₃ heterocycloalkyl, two or moreof R^(e), R^(f), and R^(g) optionally being joined by one or more bondsto form one or more optionally substituted carbocycles and/orheterocycles, and R^(5′) may in addition be absent; R¹ is selected fromhalogen and OSO₂R^(o); X⁴ is CH; R⁸, R⁹, R¹⁰, and R¹¹ are eachindependently selected from H, R^(h), NO₂, halogen, N(R^(h))R^(i),N(R^(h))C(O)R^(i), OC(O)N(R^(h))R^(i), OC(O)OR^(h), C(O)R^(h), SR^(h),OR^(h), and O(CH₂)_(bb)N(R¹⁰¹)(R¹⁰¹), wherein R¹⁰¹ and R¹⁰² areindependently H or unsubstituted C₁₋₃ alkyl, bb is an integer from 1 to20, and R^(h) and R^(i) are independently selected from H and optionallysubstituted C₁₋₁₅ alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅ cycloalkyl, C₁₋₁₅heterocycloalkyl, C₆₋₁₅ aryl, or C₁₋₁₅ heteroaryl, provided that atleast one of R⁸, R⁹, R¹⁰, and R¹¹ comprises at least one water-solublegroup.

In another embodiment, this invention relates to a compound of formula(Ib) wherein R², R⁴, and R^(4′) are H, R⁵ is selected from OH, SH, NH₂,N₃, NO₂, NO, halogen, SR^(e), S(O)R^(e), S(O)₂R^(e), S(O)OR^(e),S(O)₂OR^(e), OS(O)R^(e), OS(O)₂R^(e), OS(O)OR^(e), OS(O)₂OR^(e), OR^(e),NHR^(e), N(R^(e))R^(f), ⁺N(R^(e))(R^(f))R^(g), P(O)(OR^(e))(OR^(f)),OP(O)(OR^(e))(OR^(f)), SiR^(e)R^(f)R^(g), OC(O)R^(e), N(R^(e))C(O)R^(f),N(R^(e))C(O)OR^(f), and N(R^(e))C(O)N(R^(f))R^(g), wherein R^(e), R^(f),and R^(g) are independently selected from H and optionally substitutedC₁₋₃ alkyl, C₁₋₃ heteroalkyl, C₃ cycloalkyl, or C₁₋₃ heterocycloalkyl,two or more of R^(e), R^(f), and R^(g) optionally being joined by one ormore bonds to form one or more optionally substituted carbocycles and/orheterocycles; X² is CR¹⁴; R¹ is selected from halogen and OSO₂R^(o); X⁴is CH; a is 0; R⁸, R⁹, R¹⁰, and R¹¹ are each independently selected fromH, R^(h), NO₂, halogen, N(R^(h))R^(i), N(R^(h))C(O)R^(i),OC(O)N(R^(h))R^(i), OC(O)OR^(h), C(O)R^(h), SR^(h), OR^(h), andO(CH₂)_(bb)N(R¹⁰⁰)(R¹⁰²), wherein R¹⁰¹ and R¹⁰² are independently H orunsubstituted C₁₋₃ alkyl, bb is an integer from 1 to 20, and R^(h) andR^(i) are independently selected from H and optionally substituted C₁₋₁₅alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅ cycloalkyl, C₁₋₁₅ heterocycloalkyl,C₆₋₁₅ aryl, or C₁₋₁₅ heteroaryl, provided that at least one of R⁸, R⁹,R¹⁰, and R¹¹ comprises at least one water-soluble group.

In another embodiment, this invention relates to a compound of formula(I) or (II) wherein R², R³, R^(3′), R⁴, and R^(4′) are H, R⁵ and R^(5′)are independently selected from OH, SH, NH₂, N₃, NO₂, NO, halogen,SR^(e), S(O)R^(e), S(O)₂R^(e), S(O)OR^(e), S(O)₂OR, OS(O)R^(e),OS(O)₂R^(e), OS(O)OR, OS(O)₂OR^(e), OR^(e), NHR^(e), N(R^(e))R^(f),⁺N(R^(e))(R^(f))R^(g), P(O)(OR^(e))(OR^(f)), OP(O)(OR^(e))(OR^(f)),SiR^(e)R^(f)R^(g), OC(O)R^(e), N(R^(e))C(O)R^(f), N(R^(e))C(O)OR^(f),and N(R^(e))C(O)N(R^(f))R^(g), wherein R^(e), R^(f), and R^(g) areindependently selected from H and optionally substituted C₁₋₃ alkyl,C₁₋₃ heteroalkyl, C₃ cycloalkyl, or C₁₋₃ heterocycloalkyl, two or moreof R^(e), R^(f), and R^(g) optionally being joined by one or more bondsto form one or more optionally substituted carbocycles and/orheterocycles, and R^(5′) may in addition be absent; R¹ is selected fromhalogen and OSO₂R^(o); X⁴ is CH; X³ is NH; X⁵ is O; R⁸, R⁹, R¹⁰, and R¹¹are each independently selected from H, R^(h), NO₂, halogen,N(R^(h))R^(i), N(R^(h))C(O)R^(i), OC(O)N(R^(h))R^(i), OC(O)OR^(h),C(O)R^(h), SR^(h), OR^(b), and O(CH₂)_(bb)N(R¹⁰¹)(R¹⁰²), wherein R¹⁰¹and R¹⁰² are independently H or unsubstituted C₁₋₃ alkyl, bb is aninteger from 1 to 20, and R^(h) and R^(i) are independently selectedfrom H and optionally substituted C₁₋₁₅ alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅cycloalkyl, C₁₋₁₅ heterocycloalkyl, C₆₋₁₅ aryl, or C₁₋₁₅ heteroaryl,provided that at least one of R⁸, R⁹, R¹⁰, and R¹¹ comprises at leastone water-soluble group.

In another embodiment, this invention relates to a compound of formula(Ib) wherein R², R⁴, and R^(4′) are H, R⁵ is selected from OH, SH, NH₂,N₃, NO₂, NO, halogen, SR^(e), S(O)R^(e), S(O)₂R^(e), S(O)OR^(e),S(O)₂OR^(e), OS(O)R^(e), OS(O)₂R^(e), OS(O)OR^(e), OS(O)₂OR^(e), OR^(e),NHR^(e), N(R^(e))R^(f), ⁺N(R^(e))(R^(f))R^(g), P(O)(OR^(e))(OR^(f)),OP(O)(OR^(e))(OR^(f)), SiR^(e)R^(f)R^(g), OC(O)R^(e), N(R^(e))C(O)R^(f),N(R^(e))C(O)OR^(f), and N(R^(e))C(O)N(R^(f))R^(g), wherein R^(e), R^(f),and R^(g) are independently selected from H and optionally substitutedC₁₋₃ alkyl, C₁₋₃ heteroalkyl, C₃ cycloalkyl, or C₁₋₃ heterocycloalkyl,two or more of R^(e), R^(f), and R^(g) optionally being joined by one ormore bonds to form one or more optionally substituted carbocycles and/orheterocycles; R¹ is selected from halogen and OSO₂R^(o); X⁴ is CH; X² isCR¹⁴; a is 0; X³ is NH; X⁵ is O; R⁸, R⁹, R¹⁰, and R¹¹ are eachindependently selected from H, R^(h), NO₂, halogen, N(R^(h))R^(i),N(R^(h))C(O)R^(i), OC(O)N(R^(h))R^(i), OC(O)OR^(h), C(O)R^(h), SR^(h),OR^(h), and O(CH₂)_(bb)N(R¹⁰¹)(R¹⁰²), wherein R¹⁰¹ and R¹⁰² areindependently H or unsubstituted C₁₋₃ alkyl, bb is an integer from 1 to20, and R^(h) and R^(i) are independently selected from H and optionallysubstituted C₁₋₁₅ alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅ cycloalkyl, C₁₋₁₅heterocycloalkyl, C₆₋₁₅ aryl, or C₁₋₁₅ heteroaryl, provided that atleast one of R⁸, R⁹, R¹⁰, and R¹¹ comprises at least one water-solublegroup.

In another embodiment, this invention relates to a compound of formula(I) or (II) wherein R², R³, R^(3′), R⁴, and R^(4′) are H, R⁵ and R^(5′)are independently selected from R^(e1), C(O)N(R^(e2))R^(f2),C(O)OR^(e2), OC(O)N(R^(e2))R^(f2), OC(O)OR^(e2), wherein R^(e1) isoptionally substituted C₁₋₃ alkyl and R^(e2) and R^(f2) areindependently selected from H and optionally substituted C₁₋₃ alkyl, andR^(5′) may in addition be absent; R⁶ and R^(6′) are independentlyselected from H and optionally substituted C₁₋₆ alkyl, unsubstitutedC₁₋₆ heteroalkyl, cyano, or C₁₋₆ alkoxy, and R^(6′) may in addition beabsent; R⁷ and R^(7′) are independently selected from H and optionallysubstituted C₁₋₆ alkyl or unsubstituted C₁₋₆ heteroalkyl, and R^(7′) mayin addition be absent; R¹ is selected from halogen and OSO₂R^(o); X⁴ isCH; X² is C(R¹⁴)(R^(14′)); R¹⁴ and R^(14′) are independently H oroptionally substituted C₁₋₆ alkyl, and R^(14′) may in addition beabsent; X³ is NH; X⁵ is O; R⁸, R⁹, R¹⁰, and R¹¹ are each independentlyselected from H, R^(h), NO₂, halogen, N(R^(h))R^(i), N(R^(h))C(O)R^(i),OC(O)N(R^(h))R^(i), OC(O)OR^(h), C(O)R^(h), SR^(h), and OR^(h), whereinR^(h) and R^(i) are independently selected from H and optionallysubstituted C₁₋₁₅ alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅ cycloalkyl, C₁₋₁₅heterocycloalkyl, C₆₋₁₅ aryl, or C₁₋₁₅ heteroaryl, provided that atleast one of R⁸, R⁹, R¹⁰, and R¹¹ comprises at least one water-solublegroup and that none of R⁵, R⁹, R¹⁰, and R¹¹ is O(CH₂)_(bb)N(R¹⁰¹)(R¹⁰²),wherein R¹⁰⁰ and R¹⁰² are independently H or unsubstituted alkyl and bbis an integer from 1 to 20.

In another embodiment, this invention relates to a compound of formula(Ib) wherein R², R⁴, and R^(4′) are H, R⁵ is selected from R^(e1),C(O)N(R^(e2))R^(f2), C(O)OR^(e2), OC(O)N(R^(e2))R^(f2), OC(O)OR^(e2),wherein R^(e1) is optionally substituted C₁₋₃ alkyl and R^(e2) andR^(f2) are independently selected from H and optionally substituted C₁₋₃alkyl; R⁶ is selected from H and optionally substituted C₁₋₆ alkyl,unsubstituted C₁₋₆ heteroalkyl, cyano, or C₁₋₆ alkoxy; R⁷ is selectedfrom H and optionally substituted C₁₋₆ alkyl or unsubstituted C₁₋₆heteroalkyl; R¹ is selected from halogen and OSO₂R^(o); X⁴ is CH; X² isCR¹⁴; R¹⁴ is H or optionally substituted C₁₋₆ alkyl; a is 0; X³ is NH;X⁵ is O; R⁸, R⁹, R¹⁰, and R¹¹ are each independently selected from H,R^(h), NO₂, halogen, N(R^(h))R^(i), N(R^(h))C(O)R^(i),OC(O)N(R^(h))R^(i), OC(O)OR^(h), C(O)R^(h), SR^(h), and OR^(h), whereinR^(h) and R^(i) are independently selected from H and optionallysubstituted C₁₋₁₅ alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅ cycloalkyl, C₁₋₁₅heterocycloalkyl, C₆₋₁₅ aryl, or C₁₋₁₅ heteroaryl, provided that atleast one of R⁸, R⁹, R¹⁰, and R¹¹ comprises at least one water-solublegroup and that none of R⁸, R⁹, R¹⁰, and R¹¹ is O(CH₂)_(bb)N(R¹⁰¹)(R¹⁰²),wherein R¹⁰⁰ and R¹⁰² are independently H or unsubstituted alkyl and bbis an integer from 1 to 20.

In another embodiment, this invention relates to a compound of formula(I) or (II) wherein R², R³, R^(3′), R⁴, and R^(4′) are H, R⁵ and R^(5′)are independently selected from R^(e1), C(O)N(R^(e2))R^(f2),C(O)OR^(e2), OC(O)N(R^(e2))R^(f2), OC(O)OR^(e2), wherein R^(e1) issubstituted methyl or optionally substituted C₂₋₃ alkyl, and R^(e2) andR^(f2) are independently selected from H and optionally substituted C₁₋₃alkyl; R⁶ and R^(6′) are independently selected from H and optionallysubstituted C₁₋₆ alkyl, unsubstituted C₁₋₆ heteroalkyl, cyano, or C₁₋₆alkoxy, and R^(6′) may in addition be absent; R⁷ and R^(7′) areindependently selected from H and optionally substituted C₁₋₆ alkyl orunsubstituted C₁₋₆ heteroalkyl, and R^(7′) may in addition be absent; R¹is selected from halogen and OSO₂R^(o); X⁴ is CH; X² is C(R¹⁴)(R^(14′));R¹⁴ and R^(14′) are independently H or optionally substituted C₁₋₆alkyl, and R^(14′) may in addition be absent; X³ is NH; X^(s) is O; R⁸,R⁹, R¹⁰, and R¹¹ are each independently selected from H, R^(h), NO₂,halogen, N(R^(h))R^(i), N(R^(h))C(O)R^(i), OC(O)N(R^(h))R^(i),OC(O)OR^(h), C(O)R^(h), SR^(h), and OR^(h), wherein R^(h) and R^(i) areindependently selected from H and optionally substituted C₁₋₁₅ alkyl,C₁₋₁₅ heteroalkyl, C₃₋₁₅ cycloalkyl, C₁₋₁₅ heterocycloalkyl, C₆₋₁₅ aryl,or C₁₋₁₅ heteroaryl, provided that at least one of R⁸, R⁹, R¹⁰, and R¹¹is O(CH₂)_(bb)N(R¹⁰¹)(R¹⁰²), wherein R¹⁰¹ and R¹⁰² are independently Hor unsubstituted C₁₋₃ alkyl and bb is an integer from 1 to 20.

In another embodiment, this invention relates to a compound of formula(Ib) wherein R², R⁴, and R^(4′) are H, R⁵ is selected from R^(e1),C(O)N(R^(e2))R^(f2), C(O)OR^(e2), OC(O)N(R^(e2))R^(f2), OC(O)OR^(e2),wherein R^(e1) is substituted methyl or optionally substituted C₂₋₃alkyl, and R^(e2) and R^(f2) are independently selected from H andoptionally substituted C₁₋₃ alkyl; R⁶ is selected from H and optionallysubstituted C₁₋₆ alkyl, unsubstituted C₁₋₆ heteroalkyl, cyano, or C₁₋₆alkoxy; R⁷ is selected from H and optionally substituted C₁₋₆ alkyl orunsubstituted C₁₋₆ heteroalkyl; R¹ is selected from halogen andOSO₂R^(o); X⁴ is CH; X² is CR¹⁴; R¹⁴ is H or optionally substituted C₁₋₆alkyl; a is 0; X³ is NH; X⁵ is O; R⁸, R⁹, R¹⁰ and R¹¹ are eachindependently selected from H, R^(h), NO₂, halogen, N(R^(h))R^(i),N(R^(h))C(O)R^(i), OC(O)N(R^(h))R^(i), OC(O)OR^(h), C(O)R^(h), SR^(h),and OR^(h), wherein R^(h) and R^(i) are independently selected from Hand optionally substituted C₁₋₁₅ alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅cycloalkyl, C₁₋₁₅ heterocycloalkyl, C₆₋₁₅ aryl, or C₁₋₁₅ heteroaryl,provided that at least one of R⁸, R⁹, R¹⁰, and R¹¹ isO(CH₂)_(bb)N(R¹⁰¹)(R¹⁰²), wherein R¹⁰¹ and R¹⁰² are independently H orunsubstituted C₁₋₃ alkyl and bb is an integer from 1 to 20.

In another embodiment, this invention relates to a compound of formula(I) or (II) wherein R², R³, R^(3′), R⁴, and R^(4′) are H, R⁵ and R^(5′)are independently selected from R^(e1), C(O)N(R^(e2))R^(f2), C(O)OR^(e2)OC(O)N(R^(e2))R^(f2), OC(O)OR^(e2), wherein R^(e1) is optionallysubstituted C₁₋₃ alkyl and R^(e2) and R^(f2) are independently selectedfrom H and optionally substituted C₁₋₃ alkyl; R⁶ and R^(6′) areindependently selected from H and optionally substituted C₁₋₆ alkyl,unsubstituted C₁₋₆ heteroalkyl, cyano, or C₁₋₆ alkoxy, and R^(6′) may inaddition be absent; R⁷ and R^(7′) are independently selected from H andoptionally substituted C₁₋₆ alkyl or unsubstituted C₁₋₆ heteroalkyl, andR^(7′) may in addition be absent; X² is C(R¹⁴)(R^(14′)); R¹⁴ and R^(14′)are independently H or optionally substituted C₁₋₆ alkyl, and R^(14′)may in addition be absent; R¹ is selected from halogen and OSO₂R^(o); X⁴is CH; X³ is NH; X⁵ is O; R⁸, R⁹, R¹⁰, and R¹¹ are each independentlyselected from H, R^(h), NO₂, halogen, N(R^(h))R^(i), N(R^(h))C(O)R^(i),OC(O)N(R^(h))R^(i), OC(O)OR^(h), C(O)R^(h), SR^(h), and OR^(h), whereinR^(h) and R^(i) are independently selected from H and optionallysubstituted C₁₋₁₅ alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅ cycloalkyl, C₁₋₁₅heterocycloalkyl, C₆₋₁₅ aryl, or C₁₋₁₅ heteroaryl, provided that none ofR⁸, R⁹, R¹⁰, and R¹¹ comprises a water-soluble group.

In another embodiment, this invention relates to a compound of formula(Ib) wherein R², R⁴, and R^(4′) are H, R⁵ is selected from R^(e1),C(O)N(R^(e2))R^(f2), C(O)OR^(e2), OC(O)N(R^(e2))R^(f), OC(O)OR^(e2),wherein R^(e1) is optionally substituted C₁₋₃ alkyl and R^(e2) andR^(f2) are independently selected from H and optionally substituted C₁₋₃alkyl; R⁶ is selected from H and optionally substituted C₁₋₆ alkyl,unsubstituted C₁₋₆ heteroalkyl, cyano, or C₁₋₆ alkoxy; R⁷ is selectedfrom H and optionally substituted C₁₋₆ alkyl or unsubstituted C₁₋₆heteroalkyl; X² is CR¹⁴; R¹⁴ is H or optionally substituted C₁₋₆ alkyl;a is 0; R¹ is selected from halogen and OSO₂R^(o); X⁴ is CH; X³ is NH;X⁵ is O; R⁸, R⁹, R¹⁰, and R¹¹ are each independently selected from H,R^(h), NO₂, halogen, N(R^(h))R^(i), N(R^(h))C(O)R^(i),OC(O)N(R^(h))R^(i), OC(O)OR^(h), C(O)R^(h), SR^(h), and OR^(h), whereinR^(h) and R^(i) are independently selected from H and optionallysubstituted C₁₋₁₅ alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅ cycloalkyl, C₁₋₁₅heterocycloalkyl, C₆₋₁₅ aryl, or C₁₋₁₅ heteroaryl, provided that none ofR⁸, R⁹, R¹⁰, and R¹¹ comprises a water-soluble group.

In one embodiment, a compound of this invention is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of this invention is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of this invention is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of this invention is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of this invention is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of this invention is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of this invention is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of this invention is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of this invention is represented by

or by an isomer, or by a mixture of isomers.

In one embodiment, a compound of this invention is represented by

or by an isomer, or by a mixture of isomers.

In one embodiment, a compound of this invention is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of this invention is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of this invention is represented by

or by an isomer, or by a mixture of isomers.Conjugates and Linker-Agent Conjugates

In another aspect, this invention relates to a conjugate of a compoundof formula (I) or (II) that can be converted in vivo and in one or moresteps to a compound of formula (I) or (II), respectively.

These conjugates may favorably affect the pharmacokinetic properties andother characteristics of a compound of formula (I) or (II). In oneembodiment, this invention relates to a conjugate comprising a compoundof formula (I) or (II) conjugated to at least one promoiety, i.e., amoiety that can be removed in vivo to release a compound of formula (I)or (II). In another embodiment, this invention relates to a conjugatecomprising a compound of formula (I) or (II) conjugated to onepromoiety.

In a further embodiment, this invention relates to a compound of formula(III):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinV² is either absent or a functional moiety;each L² is independently absent or a linking group linking V² to L;each L is independently absent or a linking group linking L² to one ormore V¹ and/or Y;each V¹ is independently absent or a conditionally-cleavable orconditionally-transformable moiety, which can be cleaved or transformedby a chemical, photochemical, physical, biological, or enzymaticprocess;each Y is independently absent or a self-eliminating spacer system whichis comprised of 1 or more self-elimination spacers and is linked to V¹,optionally L, and one or more Z;each p and q are numbers representing a degree of branching and are eachindependently a positive integer;z is a positive integer equal to or smaller than the total number ofattachment sites for Z;each Z is independently a compound of formula (I) or (II) as definedhereinabove wherein one or more of X¹, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ mayoptionally in addition be substituted by a substituent of formula (V):

wherein each V^(2′), L^(2′), L′, V^(1′), Y′, Z′, p′, q′, and z′ has thesame meaning as defined for V², L², L, V¹, Y, Z, p, q, and z,respectively, the one or more substituents of formula (V) beingindependently connected to one or more of X¹, R⁶, R⁷, R⁸, R⁹, R¹⁰, andR¹¹ via Y′, each Z being independently connected to Y through either X¹or an atom in R⁶, R⁷, R⁸, R⁹, R¹⁰, or R¹¹.

It should be understood from formula (III) that L can either beconnected to V¹ and/or to Y. If L is connected to Y, this means thatboth V¹ and L, as well as one or more Z, are connected to Y. If L isconnected to V¹, this means that V¹ and one or more Z are connected toY. L may also be connected to both V¹ and Y at the same time. If Y isabsent, L is connected to V¹ or, if V¹ is absent, L is directlyconnected to Z.

The V²(-L²-L(-(V¹—Y))_(p))_(q)(Z)_(z-1) and one or moreV^(2′)(-L^(2′)-L′(-(V^(1′)—Y′))_(p′))_(q′)(Z′)_(z′-1) moieties connectedto a compound of formula (I) or (II) are herein referred to aspromoieties.

The present invention also relates to a compound of formula (IV):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinRM is a reactive moiety and L, V¹, Y, Z, p, and z are as defined above,except that L is now linking RM to one or more V¹ and/or Y, and V¹, Y,and Z may contain protecting groups, and the one or more V^(2′)-L^(2′)moieties optionally present in Z as defined hereinabove may optionallyand independently be replaced by RM′, which is a reactive moiety, andwherein, if there is more than 1 reactive moiety in (IV), some or allreactive moieties are the same or different. These linker-agentconjugates of formula (IV) may or may not be considered intermediatesfor compounds of formula (III).

The RM-L(-(V¹—Y))_(p)(Z)_(z-1) and one or moreRM′-L′(-(V^(1′)—Y′))_(p′)(Z′)_(z′-1) moieties connected to a compound offormula (I) or (II) are herein referred to as promoieties.

It should be understood that this invention relates to enantiomericallypure and/or diastereomerically pure compounds of formulae (III) and (IV)as well as to enantiomeric and/or diastereomeric mixtures of compoundsof formulae (III) and (IV).

When a compound of formula (III) or (IV) contains attachment sites for Zthat are not coupled to Z, for instance as a consequence of anincomplete coupling reaction during synthesis, these attachment sitesare considered to be attached to H, OH, or a leaving group instead. Ifall of said attachment sites are connected to Z, then z equals thenumber of said attachment sites; otherwise, z is lower. Compounds ofthis invention may exist as a mixture, wherein each component of themixture has a different z value. For example, the compound may exist asa mixture of two separate compounds, one compound wherein z is 4 andanother compound wherein z is 3. Furthermore, for a given z, thecompound may exist as a mixture of (constitutional) isomers as Z may beconnected to distinct sets of attachment sites.

For reasons of clarity, when referring to the connections of one firstmoiety to other moieties within formula (III) or (IV), in general onlythose said other moieties are mentioned that are directly next to saidfirst moiety in formula (III) or (IV). It should be understood that ifone of said other moieties is not present, said first moiety is actuallyconnected to the moiety first in line that is present, unless explicitlystated otherwise. For example, if it is stated that “V¹ is cleaved fromY”, this phrase actually means “V¹ is cleaved from Y, or from Z if Y isabsent” and should be read as “V¹ is cleaved from Z” when reference ismade to a compound lacking Y.

In a compound of formula (III) or (IV), a compound of formula (I) or(II) may be conjugated to a promoiety through its water-soluble group.In this way, the water-soluble group may contribute less to the watersolubility of the compound of formula (III) or (IV), but may contributeagain to the water solubility of Z upon removal of said promoiety.

In this document, whenever V², L², L, V¹, Y, Z, RM, p, q, or z ismentioned, it should be understood that the same can apply for eachV^(2′), L^(2′), L′, V^(1′), Y′, Z′, RM′, p′, q′, or z′, respectively.

The V¹ Moiety

In a compound of formula (III) or (IV), the V¹ moiety can be a groupthat is conditionally cleavable or transformable. In other words, it isdesigned to be transformed and/or cleaved from Y by a chemical,photochemical, physical, biological, or enzymatic process upon beingbrought in or under a certain condition. This condition may for examplebe bringing a compound of the invention in an aqueous environment, whichleads to hydrolysis of V¹, or bringing a compound of the invention in anenvironment that contains an enzyme that recognizes and cleaves V¹, orbringing a compound of the invention under reducing conditions, whichleads to reduction and/or removal of V¹, or bringing a compound of theinvention under oxidizing conditions, which leads to oxidation and/orremoval of V¹, or bringing a compound of the invention in contact withradiation, e.g., UV light, which leads to transformation and/orcleavage, or bringing a compound of the invention in contact with heat,which leads to transformation and/or cleavage, or bringing a compound ofthe invention under reduced pressure, which leads to transformation,e.g., a retrocycloaddition, and/or cleavage, or bringing a compound ofthe invention under elevated or high pressure, which leads totransformation and/or cleavage. This condition may be met afteradministrating a compound of this invention to an animal, e.g., amammal, for example a human: the condition may be met when the compoundlocalizes to for example a specific organ, tissue, cell, subcellulartarget, or microbial target, for example by the presence of internalfactors (e.g., target-specific enzymes or hypoxia) or application ofexternal factors (e.g., radiation, magnetic fields) or the condition mayalready be met directly upon administration (e.g., ubiquitous enzymes).

In general, transformation of V¹ will directly or indirectly lead tocleavage of V¹ from Y. Alternatively, transformation of V¹ may lead toformation of a V¹—Y moiety which is a self-immolative linker. Forexample, oxidation of V¹ being a hydrogen atom to a hydroxyl group maylead to a para- or ortho-hydroxybenzyl moiety that self-eliminates.

Alternatively again, V¹ may be absent. In this instance, the promoietyis intended to be non-removable from Z and the whole promoiety or a partthereof (due to degradation of a compound of formula (III) or (IV) atone or more other sites in the molecule) will stay connected to the oneor more moieties Z.

A compound of this invention may contain more than one V¹ moiety perpromoiety (p and/or q>1). These V¹ moieties may or may not be the sameand may or may not require the same conditions for transformation and/orcleavage.

In one aspect of this invention, a conjugate is used to target one ormore moieties Z to target cells. In this instance, a V¹ moiety may forexample contain a substrate molecule that is cleaved by an enzymepresent in the vicinity of the target cells or inside the target cells,for example tumor cells. V¹ can for example contain a substrate that iscleaved by an enzyme present at elevated levels in the vicinity of orinside the target cells as compared to other parts of the body, or by anenzyme that is present only in the vicinity of or inside the targetcells.

It is important to recognize that if target cell specificity is achievedsolely based upon the selective transformation and/or cleavage of saidV¹ at the target site, the condition causing the cleavage shouldpreferably, at least to a certain degree, be target cell-specific,whereas the presence of another target-specific moiety in the compoundof the invention, for instance in a V² moiety, reduces or takes awaythis requirement. For example, when V² causes selective internalizationinto a target cell, an enzyme also present in other cells may transformand/or cleave V¹. In one embodiment, transformation and/or cleavage ofV¹ occur intracellularly. In another embodiment, transformation and/orcleavage of V¹ occur extracellularly.

In one embodiment, V¹ contains a di-, tri-, tetra-, or oligopeptidewhich consists of an amino acid sequence recognized by a proteolyticenzyme, for example plasmin, a cathepsin, cathepsin B, prostate-specificantigen (PSA), urokinase-type plasminogen activator (u-PA), or a memberof the family of matrix metalloproteinases, present in the vicinity ofor inside the target cells, for example tumor cells. In one embodiment,V¹ is a peptide. In another embodiment, V¹ is a dipeptide. In anotherembodiment, V¹ is a tripeptide. In another embodiment, V¹ is atetrapeptide. In yet another embodiment, V¹ is a peptidomimetic.

In another embodiment, V¹ contains a β-glucuronide that is recognized byβ-glucuronidase present in the vicinity of or inside tumor cells.

In one embodiment, V¹ contains a substrate for an enzyme.

In one embodiment, V¹ contains a substrate for an extracellular enzyme.

In another embodiment, V¹ contains a substrate for an intracellularenzyme.

In yet another embodiment, V¹ contains a substrate for a lysosomalenzyme.

In yet another embodiment, V¹ contains a substrate for the serineprotease plasmin.

In yet another embodiment, V¹ contains a substrate for one or more ofthe cathepsins, for example cathepsin B.

In yet another embodiment, V¹ contains a substrate for a galactosidase.

When V¹ is cleaved extracellularly, the one or more Z moieties may bereleased extracellularly. This may provide the advantage that these Zmoieties are not only able to affect the cell(s) directly surroundingthe site of activation (e.g., target-positive cells), but also cellssomewhat further away from the site of activation (e.g., target-negativecells) due to diffusion (bystander effect).

An enzyme to cleave V¹ can also be transported to the vicinity of orinside target cells or target tissue via for example antibody-directedenzyme prodrug therapy (ADEPT), polymer-directed enzyme prodrug therapy(PDEPT) or macromolecular-directed enzyme prodrug therapy (MDEPT),virus-directed enzyme prodrug therapy (VDEPT), or gene-directed enzymeprodrug therapy (GDEPT). In one embodiment, transformation and/orcleavage of V¹ occur through an enzyme linked to an antibody.

In again another embodiment V¹ contains a moiety, for example a nitro(hetero)aromatic moiety, that can be transformed and/or cleaved byreduction under hypoxic conditions or by reduction by a nitroreductase.After reduction of the nitro group and cleavage of the resulting moiety,elimination of the spacer system Y, if present, leads to release of oneor more moieties Z.

In one embodiment the invention relates to a conjugate wherein V¹ is adipeptide, tripeptide, tetrapeptide, or oligopeptide moiety comprised ofnatural L amino acids, unnatural D amino acids, or synthetic aminoacids, or a peptidomimetic, or any combination thereof.

In another embodiment the invention relates to a compound wherein V¹comprises a tripeptide. The tripeptide may be linked via its C-terminusto Y. In one embodiment, the C-terminal amino acid residue of thetripeptide is selected from arginine, citrulline, and lysine, the middleamino acid residue of the tripeptide is selected from alanine, valine,leucine, isoleucine, methionine, phenylalanine, cyclohexylglycine,tryptophan, and proline, and the N-terminal amino acid residue of thetripeptide is selected from any natural or unnatural amino acid.

In another embodiment the invention relates to a compound wherein V¹comprises a dipeptide. The dipeptide may be linked via its C-terminus toY. In one embodiment, the C-terminal amino acid residue of the dipeptideis selected from alanine, arginine, citrulline, and lysine, and theN-terminal amino acid residue of the dipeptide is selected from anynatural or unnatural amino acid.

In one embodiment, when the α-amino group of the N-terminal amino acidof V¹ is not coupled to L, this amino acid may be functionalized with asuitable blocking group coupled to the α-amino group or may be anunnatural amino acid such that undesired premature degradation of V¹ byfor example ubiquitous enzymes or exopeptidases is prevented.

In a further embodiment V¹ is selected from D-alanylphenylalanyllysine,D-valylleucyllysine, D-alanylleucyllysine, D-valylphenylalanyllysine,D-valyltryptophanyllysine, D-alanyltryptophanyllysine,alanylphenylalanyllysine, valylleucyllysine, alanylleucyllysine,valylphenylalanyllysine, valyltryptophanyllysine,alanyltryptophanyllysine, D-alanylphenylalanylcitrulline,D-valylleucylcitrulline, D-alanylleucylcitrulline,D-valylphenylalanylcitrulline, D-valyltryptophanylcitrulline,D-alanyltryptophanylcitrulline, alanylphenylalanylcitrulline,valylleucylcitrulline, alanylleucylcitrul line,valylphenylalanylcitrulline, valyltryptophanylcitrulline, andalanyltryptophanylcitrulline.

In yet another embodiment, V¹ is selected from phenylalanyllysine,valyllysine, valylalanine, D-phenylalanylphenylalanyllysine,phenylalanylphenylalanyllysine, glycylphenylalanyllysine, alanyllysine,valylcitrulline, N-methylvalylcitrulline, phenylalanylcitrulline,isoleucylcitrulline, tryptophanyllysine, tryptophanylcitrulline,phenylalanylarginine, phenylalanylalanine,glycylphenylalanylleucylglycine, alanylleucylalanylleucine,alanylarginylarginine, phenylalanyl-N⁹-tosylarginine,phenylalanyl-N⁹-nitroarginine, leucyllysine, leucylcitrulline, andphenylalanyl-O-benzoylthreonine.

In a further embodiment, V¹ is selected from phenylalanyllysine,valyllysine, and valylcitrulline.

Therefore, in one embodiment this invention relates to a compoundwherein V¹ contains a substrate that can be cleaved by a proteolyticenzyme, plasmin, a cathepsin, cathepsin B, β-glucuronidase, agalactosidase, prostate-specific antigen (PSA), urokinase-typeplasminogen activator (u-PA), a member of the family of matrixmetalloproteinases, or an enzyme localized by means of directed enzymeprodrug therapy, such as ADEPT, VDEPT, MDEPT, GDEPT, or PDEPT, orwherein V¹ contains a moiety that can be cleaved or transformed throughreduction under hypoxic conditions, through reduction by anitroreductase, or through oxidation.

In another aspect of this invention, a conjugate of this invention isused to (also) improve the (pharmacokinetic) properties of Z. When apromoiety does not need to be selectively removed at a target site, V¹of said promoiety may for example be or contain a group that is cleavedby ubiquitous enzymes, e.g., esterases that are present in thecirculation or intracellular enzymes, such as for example proteases andphosphatases, by pH-controlled intramolecular cyclization, or byacid-catalyzed, base-catalyzed, or non-catalyzed hydrolysis, or V¹ mayfor example be or contain a disulfide or form a disulfide with aneighboring moiety. V¹ may therefore, optionally together with theconnecting atom(s) of L and/or Y, for example form a carbonate,carbamate, ureum, ester, amide, imine, hydrazone, oxime, disulfide,acetal, or ketal group that can be cleaved in vivo. This means that V¹,optionally together with the connecting atom(s) of L and/or Y, can forexample also represent —OC(O)—, —C(O)O—, —OC(O)O—, —OC(O)N(R^(v))—,—N(R^(v))C(O)—, —C(O)N(R^(v))—, —N(R^(v))C(O)O—, —N(R^(v))C(O)N(R^(w))—,—C(O)—, —OC(R^(v))(R^(w))—, —C(R^(v))(R^(w))O—, —OC(R^(v))(R^(w))O—,—C(R^(v))(R^(w))—, —S—, —S—S—, —C═, ═C—, —N═, ═N—, —C═N—, —N═C—, —O—N═,═N—O—, —C═N—O—, —O—N═C—, —N(R^(v))—N═, ═N—N(R^(v))—, —N(R^(v))—N═C—, or—C═N—N(R^(v))—, wherein R^(v) and R^(w) are independently selected fromH and optionally substituted C₁₋₁₀ alkyl or C₆₋₁₀ aryl, R^(v) and R^(w)optionally being joined by one or more bonds to form one or moreoptionally substituted carbocycles and/or heterocycles.

If V¹ or V¹—Y represents a whole promoiety or L is connected to Y andnot to V¹, V¹ may in this case for example be selected fromR^(p)—[O(R^(p′)O)P(O)]_(pp)—, R^(p)—C(O)—, R^(p)—OC(O)—, andR^(p)—N(R^(p))C(O)—, wherein pp is selected from 1 to 3 and each R^(p)and R^(p′) is independently selected from H and optionally substitutedC₁₋₁₅ alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅ cycloalkyl, C₁₋₁₅heterocycloalkyl, C₆₋₁₅ aryl, or C₁₋₁₅ heteroaryl, R^(p) and R^(p′)optionally being joined by a bond to form an optionally substitutedcarbocycle or heterocycle.

In one embodiment, V¹ is selected from phosphono, phenylaminocarbonyl,4-(piperidino)piperidinocarbonyl, piperazinocarbonyl, and4-methylpiperazinocarbonyl. V¹ itself may contribute to favorable(pharmacokinetic) properties of the conjugate, for example through thepresence of polar functional groups in V¹.

It should be noted that V¹, either in the form of a di-, tri-, tetra-,or oligopeptide, or in any other form, may contain protecting groups.Compounds of the invention comprising such a protected V¹ may notrelease any Z moiety when put under conditions that will transformand/or cleave the corresponding unprotected V¹. However, when saidcompounds are deprotected, such compounds will release one or more Zmoieties when put under the appropriate conditions. Compounds comprisingsuch a protected V¹ also fall under the scope of this invention. Inparticular the above can be envisioned for compounds of formula (IV).Suitable protecting groups for functional groups, in particular foramino acids, are well-known to the organic chemist and may for examplebe found in T. W. Greene, Protective Groups in Organic Synthesis, JohnWiley & Sons, New York, 1981.

Compounds of formulae (III) and (IV) can be designed to eventuallyrelease a compound of formula (I) or (II), or a compound of formula (I′)or (II′), after transformation and/or cleavage of the one or more V¹ andV^(1′) moieties. Release of a compound of formula (I) or (II), acompound of formula (I′) or (II′), or a derivative thereof, from aconjugate of this invention via another mechanism is however notexcluded from this invention.

In another aspect of this invention, a compound of formula (III)represents an intermediate for the preparation of a compound of formula(I) or (II) or another compound of formula (III). In this instance, forexample, V², L², L, and Y are absent, p, q, and z all are 1, and the V¹moiety may be a protecting group. There may or may not be one or moreV^(2′)(-L^(2′)-L′(—(V^(1′)—Y′))_(p′))_(q′)(Z′)_(z′-1) moieties, in whichV^(2′), L^(2′), L′, and Y′ may or may not be absent, and p′, q′, and z′all may or may not be 1. In one embodiment, a compound of formula (III)is a compound of formula (I) or (II) to which a V¹ moiety is attached.In another embodiment, a compound of formula (III) is a compound offormula (I) or (II) to which a V¹ moiety and aV^(2′)(-L^(2′)-L′(—(V^(1′)—Y′))_(p′))_(q′)(Z′)_(z′-1) moiety areattached. In yet another embodiment, a compound of formula (III) is acompound of formula (I) or (II) to which a V¹ moiety and a V^(1′) moietyare attached.

In one embodiment, V¹ is not a protecting group.

In another embodiment, V², L², L, and Y are absent, and p, q, and z allare 1.

In a further embodiment, V¹ is a chemically removable group.

In yet a further embodiment, V¹ is a chemically removable groupconnected to Z via X¹.

In yet another further embodiment, V¹ is a benzyl group connected to Zvia X¹.

In another embodiment, V¹ istert-butoxycarbonyl(methylamino)ethyl(methylamino)carbonyl.

In another embodiment, V¹ is4-(tert-butoxycarbonyl)piperazine-1-carbonyl.

In one embodiment, V¹ is connected to L via more than one functionalgroup on V¹.

In another embodiment, V¹ is connected to L via one functional group onV¹.

In another embodiment, V¹ is connected to L via a functional group inthe side chain of one of the natural or unnatural amino acids of V¹.

In another embodiment, the N-terminal amino acid of V¹ is connected viaits a amino group to L.

In another embodiment, V¹ is absent.

The Self-Eliminating Spacer System Y

The self-elimination spacer system Y, if present, links V¹ andoptionally L to one or more moieties Z.

A self-elimination spacer system Y may be incorporated in a conjugate ofthis invention to for example improve the properties of Z or theconjugate in general, to provide for suitable coupling chemistries,and/or to create space between V¹ and Z.

A compound of this invention may contain more than one spacer system Yper promoiety. These moieties Y may or may not be the same.

After cleavage or transformation of V¹, the left-hand side of Y maybecome unblocked or a V¹—Y self-elimination moiety is formed, whichresults in eventual release of one or more moieties Z. Theself-elimination spacer systems may for example be those described in WO02/083180 and WO 2004/043493, which are incorporated herein by referencein their entirety, as well as other self-elimination spacers known to aperson skilled in the art.

In one aspect the invention is related to compounds wherein Y isselected from

(W—)_(w)(X—)_(x)(A-)_(s)

(W—)_(w)(X—)_(x)C((A)_(s)-)_(r) or

(W—)_(w)(X—)_(x)C(D((A)_(s)-)_(d))_(r) or

(W—)_(w)(X—)_(x)C(D(E((A)_(s)-)_(e))_(d))_(r), or

(W—)_(w)(X—)_(x)C(D(E(F((A)_(s)-)_(f))_(e))_(d))_(r)

wherein

W and X are each a single-release 1,2+2n electronic cascade spacer(n≧1), being the same or different;

A is an ω-amino aminocarbonyl cyclization spacer that forms a cyclicureum derivative upon cyclization;

C, D, E, and F are each a self-eliminating multiple-release spacer orspacer system that upon activation can maximally release r, d, e, and fgroups, respectively;

s is 0 or 1;

r, d, e, and f are numbers representing degree of branching;

w and x are numbers representing degree of polymerization;

r, d, e, and fare independently an integer from 2 (included) to 24(included);

w and x are independently an integer from 0 (included) to 5 (included).

In a further aspect of the invention, the self-eliminationmultiple-release spacer systems C, D, E, and F are independentlyselected from a moiety having the formula:

whereinB is selected from NR²¹, O, and S;P is C(R²²)(R²³)Q-(W—)_(w)(X—)_(x);Q is absent or is —O—CO—;W and X are each a single-release 1,2+2n electronic cascade spacer(n≧1), being the same or different;G, H, I, J, K, L, M, N, and O are independently selected from moietieshaving the formula:

G, J, and M may in addition be selected from the group of P and hydrogenwith the proviso that if two of G, J, and M are hydrogen, the remaininggroup must be

or be

and at the same time be conjugated to

R²¹ is selected from H and optionally substituted C₁, alkyl;

R²², R²³, R²⁴, and R²⁵ are independently selected from H, OH, SH, NH₂,N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH, halogen, R^(x), SR^(x),S(O)R^(x), S(O)₂R^(x), S(O)OR^(x), S(O)₂OR^(x), OS(O)R^(x), OS(O)₂R^(x),OS(O)OR^(x), OS(O)₂OR^(x), OR^(x), NHR^(x), N(R^(x))R^(x),N(R^(x))(R^(x1))R^(x2), P(O)(OR^(x))(OR^(x1)), OP(O)(OR^(x))(OR^(x1)),C(O)R^(x), C(O)OR^(x), C(O)N(R^(x1))R^(x), OC(O)R^(x), OC(O)OR^(x),OC(O)N(R^(x))R^(x1), N(R^(x1))C(O)R^(x), N(R^(x1))C(O)OR^(x), andN(R^(x1))C(O)N(R^(x2))R^(x), wherein R^(x), R^(x1), and R^(x2) areindependently selected from H and optionally substituted C₁₋₆ alkyl,C₁₋₅ heteroalkyl, C₃₋₂₀ cycloalkyl, C₁₋₂₀ heterocycloalkyl, C₆₋₂₀ aryl,or C₁₋₂₀ heteroaryl, R^(x), R^(x1), and R^(x2) optionally being joinedby one or more bonds to form one or more optionally substitutedcarbocycles and/or heterocycles, two or more of the substituents R²¹,R²², R²³, R²⁴, and R²⁵ optionally being joined by one or more bonds toform one or more optionally substituted carbocycles and/or heterocycles;g, h, i, j, k, 1, m, n, o, h′, g′, k′, j′, n′, m′ are numbersrepresenting degree of branching and are independently 0, 1, or 2 withthe provisos that

if G=hydrogen or P, g, h, i, h′, and g′ all equal 0;

if J=hydrogen or P, j, k, 1, k′, and j′ all equal 0;

if M=hydrogen or P, m, n, o, n′, and m′ all equal 0;

if G, H, I, J, K, L, M, N, or O is

then g+g′=1, h+h′=1, i=1, j+j′=1, k+k′=1, l=1, m+m′=1, n+n′=1, or o=1,respectively;if G, H, I, J, K, L, M, N, or O is

then g+g′=2, h+h′=2, i=2, j+j′=2, k+k′=2, l=2, m+m′=2, n+n′=2, or o=1,respectively;if g′=0 and G is not hydrogen or P, then h, h′, and i equal 0 and g>0;if g=0 and G is not hydrogen or P, then g>0;if g′>0 and h′=0, then i=0 and h>0;if g′>0 and h=0, then h′>0 and i>0;if j′=0 and J is not hydrogen or P, then k, k′, and l equal 0 and j>0;if j=0 and J is not hydrogen or P, then j>0;if j′>0 and k′=0, then l=0 and k>0;if j′>0 and k=0, then k′>0 and l>0;if m′=0 and M is not hydrogen or P, then n, n′, and o equal 0 and m>0;if m=0 and M is not hydrogen or P, then m′>0;if m′>0 and n′=0, then o=0 and n>0;if m′>0 and n=0, then n′>0 and o>0;w and x are numbers of polymerization and are independently an integerfrom 0 (included) to 5 (included).

According to a further embodiment of this invention, the 1,2+2nelectronic cascade spacers W and X are independently selected from amoiety having the formula:

whereinQ′=—R³⁰C═CR³¹—, S, O, NR³¹, —R³¹C═N—, or —N═CR³¹—;B═NR³², O, S;P═C(R²⁸)(R²⁹)Q;R²⁶, R²⁷, B, and (T-)_(t)(T′-)_(t′)(T″-)_(t″), P are connected to C^(a),C^(b), C^(c), and C^(d) in such a way that B and(T-)_(t)(T′-)_(t′)(T″-)_(t″)P are connected to two adjacent carbon atomsor to C^(a) and C^(d);Q is absent or —O—CO—;t, t′, and t″ are numbers representing degree of polymerization and areindependently an integer from 0 (included) to 5 (included);T, T′, and T″ are independently selected from moieties having theformula:

R²⁶, R²⁷, R²⁸, R²⁹, R³⁰, R³¹, R³², R³³, and R³⁴ are independentlyselected from H, OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H,C(O)OH, halogen, R^(y), SR^(y), S(O)R^(y), S(O)₂R^(y), S(O)OR^(y),S(O)₂OR^(y), OS(O)R^(y), OS(O)₂R_(y), OS(O)OR^(y), OS(O)₂OR^(y), OR^(y),NHR^(y), N(R^(y))R^(y1), ⁺N(R^(y))(R^(y1))R^(y2), P(O)(OR^(y))(OR^(y1)),OP(O)(OR^(y))(OR^(y1)), C(O)R^(y), C(O)OR^(y), C(O)N(R^(y1))R^(y),OC(O)R^(y), OC(O)OR^(y), OC(O)N(R^(y))R^(y1), N(R^(y1))C(O)R^(y),N(R^(y1))C(O)OR^(y), and N(R^(y1))C(O)N(R^(y2))R^(y), wherein R^(y),R^(y1), and R^(y2) are independently selected from H and optionallysubstituted C₁₋₆ alkyl, C₁₋₆ heteroalkyl, C₃₋₂₀ cycloalkyl, C₁₋₂₀heterocycloalkyl, C₆₋₂₀ aryl, or C₁₋₂₀ heteroaryl, R^(y), R^(y1), andR^(y2) optionally being joined by one or more bonds to form one or moreoptionally substituted carbocycles and/or heterocycles, two or more ofthe substituents R²⁶, R²⁷, R²⁸, R²⁹, R³⁰R³¹, R³², R³³ and R³⁴ optionallybeing joined by one or more bonds to form one or more optionallysubstituted carbocycles and/or heterocycles.

In the formulae above, Q may be O—CO, but it may also be absent. Forexample, a compound with a benzyl ether linkage between self-eliminationspacer and the group that leaves, the oxycarbonyl function being absent(Q is absent), has been reported to undergo self-elimination¹¹.

According to a further embodiment of the invention, the ω-aminoaminocarbonyl cyclization elimination spacer A is a moiety having theformula:

whereinu is an integer of 0 or 1;R³⁵ and R³⁶ are independently selected from H and optionally substitutedC₁₋₆ alkyl;R³⁷, R³⁸, R³⁹, R⁴⁰, R⁴¹, and R⁴² are independently selected from H, OH,SH, NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH, halogen, R^(z),SR^(z), S(O)R^(z), S(O)₂R^(z), S(O)OR^(z), S(O)₂OR^(z), OS(O)R^(z),OS(O)₂R^(z), OS(O)OR^(z), OS(O)₂OR^(z), OR^(z), NHR^(z), N(R^(z))R^(z1),⁺N(R^(z))(R^(z1))R^(z2), P(O)(OR^(z))(OR^(z1)), OP(O)(OR^(z))(OR^(z1)),C(O)R^(z), C(O)OR^(z), C(O)N(R^(z1))R^(z), OC(O)R^(z), OC(O)OR^(z),OC(O)N(R^(z))R^(z1), N(R^(z1))C(O)R^(z), N(R^(z1))C(O)OR^(z), andN(R^(z1))C(O)N(R^(z2))R^(z), wherein R^(z), R^(z1), and R^(z2) areindependently selected from H and optionally substituted C₁₋₆ alkyl,C₁₋₆ heteroalkyl, C₃₋₂₀ cycloalkyl, C₁₋₂₀ heterocycloalkyl, C₆₋₂₀ aryl,or C₁₋₂₀ heteroaryl, R^(z), R^(z1), and R^(z2) optionally being joinedby one or more bonds to form one or more optionally substitutedcarbocycles and/or heterocycles, two or more of the substituents R³⁵,R³⁶, R³⁷, R³⁸, R³⁹, R⁴⁰, R⁴¹, and R⁴² optionally being joined by one ormore bonds to form one or more optionally substituted carbocycles and/orheterocycles.

In one embodiment, Y is absent.

In another embodiment, this invention relates to a compound of formula(II) or (IV) wherein X¹ is O and Y is connected to X¹ via an ω-aminoaminocarbonyl cyclization spacer being part of Y.

In one embodiment, the spacer system Y is selected from

In another embodiment, the spacer system Y is

In another embodiment, the spacer system Y is

Other examples of self-eliminating spacers include, but are not limitedto, spacers that can undergo cyclization¹², such as optionallysubstituted 4-aminobutyric acid amides, appropriately substitutedbicyclo[2.2.1] and bicyclo[2.2.2] ring systems, and2-aminophenylpropionic acid amides and “trimethyl-lock” cyclizationspacers¹³. A glycine spacer in which an amine-containing leaving groupis connected at the α-position is another useful spacer for thecompounds of the invention.¹⁴

In a conjugate of this invention, a spacer system Y may be connected tomore than one V¹ moiety. In this case, transformation and/or cleavage ofone of these V¹ moieties may trigger the release of one or more Zmoieties. When V¹ moieties that are transformed or cleaved underdifferent conditions are connected to the same Y, release of one or moreZ moieties may occur when a conjugate of this invention is brought underone of several distinct conditions. Alternatively, a spacer system Y maybe used that requires to be triggered twice or even more times in orderto self-eliminate. An example of such a self-elimination spacer is abicine spacer.¹⁵ When such a spacer is used in combination withdifferent, selectively cleavable V¹ moieties connected to said spacer,selectivity of release of Z may be increased as two different conditionsmust be met before Z is released.

The Linking Group L

The linking group L links one or more V¹ and/or Y moieties to L² or RM.Synthesis may be more straightforward when L is connected to V¹ insteadof Y and the compound may be less prone to premature degradation.Connection of L to Y may have the advantage that V¹ may be transformedand/or cleaved with more ease. Other reasons to connect L to Y may forexample be that (part of) Y remains bound to L upon cleavage of V¹,which prevents the release of reactive small molecules, or that thecompound displays improved (pharmacokinetic) properties, solubility, oraggregation behavior. L may be absent such that V¹ or Y is directlyconnected to either L² or RM. In another aspect, however, L is a linkinggroup that functionally links or spaces the one or more V¹ and/or Ymoieties and the L² or RM moiety. In a compound of formula (IV), spacingmay make the reactive moiety RM more accessible to the reaction partner,for example when the functional moiety is being coupled. In a compoundof formula (III), spacing may provide for a better accessibility of V¹,because V² is further removed, which, especially in the case ofenzymatic cleavage or transformation of V¹, may improve the rate atwhich V¹ is transformed and/or cleaved. The linking group L may be awater-soluble moiety or contain one or more water-soluble moieties, suchthat L contributes to the water solubility of a compound of formula(III) or (IV). L may also be a moiety or contain one or more moietiesthat reduce(s) aggregation of a compound of formula (III) or (IV), whichmay or may not be a moiety/moieties that also increase(s) the watersolubility of a compound of formula (III) or (IV). The linking group Lmust contain suitable functional groups at both of its ends to providefor selective coupling with the one or more V¹ and/or Y moieties and L²or RM.

In one aspect, the L moiety is a linear, branched, or dendritic moiety,so that it can optionally be connected to more than one V¹ and/or Ymoiety. Branching can occur via one or more cyclic structures or at oneor more branching atoms that may for example be carbon, nitrogen,silicon, or phosphorus.

The number of branches in L that are connected to V¹ and/or Y does notnecessarily equal the total number of branches as in the couplingreaction with V¹ and/or Y not all branches may be coupled to V¹ and/or Ymoieties due to incomplete chemical conversion. This means that L maycontain branches that are not coupled to V¹ or Y, but instead end in forexample a functional group, H, OH, or a leaving group.

Therefore, when L is branched, compounds of this invention may exist asa mixture, wherein each component of the mixture has a different pvalue. For example, the compound may exist as a mixture of two separatecompounds, one compound wherein p is 2 and another compound wherein p is3. Furthermore, for a given p, the compound may exist as a mixture of(constitutional) isomers as V¹ and/or Y may be connected to distinctsets of branches on L.

In one embodiment, L is absent.

In another embodiment, L is a linear linker.

In another embodiment, L is a linear linker built up through acycloaddition reaction between a molecule containing an azide group andone containing an acetylene group.

In another embodiment, L is a branched linker.

In another embodiment, L is a dendritic linker. The dendritic structuremay for example be built up through cycloaddition reactions betweenmolecules containing an azide group and ones containing an acetylenegroup.

In one embodiment, p is 1.

In other embodiments, p is 2 or 3 or 4 or 6 or 8 or 9.

In another embodiment, L is represented by the formula:

whereinX⁸¹ and X⁸² are each independently O, NR⁸⁵, or S;Each X⁸³ and X⁸⁴ is independently O, NR⁸⁶, or S;Each x81, x82, x83, and x84 is independently 0 or 1;p″ is a number representing a degree of branching and is an integerselected from 1 (included) to 128 (included);

-   -   p′″ is a number representing a degree of branching and is an        integer selected from 0 (included) to 127 (included);        p″+p′″≦128;        Each DD is independently H, OH, or a leaving group;        R⁸⁰ is absent or is either a dendritic, branched or unbranched        moiety and selected from optionally substituted alkylene or        polyalkylene, optionally substituted heteroalkylene or        polyheteroalkylene, optionally substituted arylene or        polyarylene, optionally substituted heteroarylene or        polyheteroarylene, optionally substituted cycloalkylene or        polycycloalkylene, optionally substituted heterocycloalkylene or        polyheterocycloalkylene, —(CH₂CH₂O)_(v)—,        -alkylene-(CH₂CH₂O)_(v)—, —(CH₂CH₂O)_(v)-alkylene-,        -alkylene-(CH₂CH₂O)_(v)-alkylene-,        -heteroalkylene-(CH₂CH₂O)_(v)—, —(CH₂CH₂O)_(v)-heteroalkylene-,        -heteroalkylene-(CH₂CH₂O)_(v)-alkylene-,        -heteroalkylene-(CH₂CH₂O), -heteroalkylene-,        -alkylene-(CH₂CH₂O), -heteroalkylene-, a dendritic structure,        and an oligopeptide, or any combination of two or more of the        above;        R⁸⁵ and R⁸⁶ are independently selected from H and C₁₋₈ alkyl;        v′ is selected from 1 (included) to 500 (included).

For example, L may be selected from optionally substituted C₁₋₁₀alkylene, C₁₋₁₀ alkylenecarbonyl, C₁₋₁₂ alkyleneoxycarbonyl, C₁₋₁₂carbonylalkylene, C₁₋₁₂ carbonylalkyleneoxycarbonyl, C₁₋₁₂alkyleneaminocarbonyl, C₁₋₁₂ alkylene(methylamino)carbonyl, and(CH₂CH₂O)_(v)-carbonyl.

In one embodiment, L is selected from

The Reactive Moiety RM and the Linking Group L²

The reactive moiety RM in a compound of formula (IV) is connected to thelinking group L and is able to react with a suitable functional group ona reaction partner.

In one embodiment of this invention, the reactive moiety RM is designedto react with a functional group on the moiety V², which results information of a compound of formula (III). In this reaction, the moietyRM is transformed into the moiety L². In another embodiment, thereactive moiety RM is designed to react with a complementary moiety insitu, e.g., in vivo, to give a compound that may or may not be acompound of formula (III).

In one aspect of this invention, the reactive moiety RM contains anelectrophilic group that reacts with a nucleophilic group on thereaction partner, for example V², e.g., a thiol group, an amino group,or a hydroxy group.

In another aspect of this invention, the reactive moiety RM contains anucleophilic group that reacts with an electrophilic group on thereaction partner, for example V², e.g., an aldehyde group.

In another aspect of the invention, the reactive moiety RM contains acycloaddition partner moiety, e.g., an alkene, a diene, a 1,3-dipole, ora 1,3-dipolarophile, that reacts with a suitable complementarycycloaddition partner moiety on the reaction partner, for example V²,e.g., a diene, an alkene, a 1,3-dipolarophile, or a 1,3-dipole.

In another aspect of the invention, the reactive moiety RM contains agroup that can be coupled with a suitable complementary group on thereaction partner, for example V², under metal-catalyzed, biocatalyzed,or enzyme-catalyzed conditions, e.g., palladium-catalyzed conditions.

In one aspect of the invention, the reactive moiety RM is, withoutlimitation,

whereinX⁸ is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,—O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl,—O—C(O)—R⁵⁰, and —O—C(O)—OR⁵⁰;X⁹ is selected from —Cl, —Br, —I, —O-mesyl, —O-triflyl, and —O-tosyl;R⁵⁰ is selected from C₁₋₁₀ alkyl and C₆₋₁₀ aryl.

In one embodiment, the moiety RM is selected from

which makes it able to react with a thiol group on the reaction partner,for example moiety V².

In another embodiment, the moiety RM is

which makes it able to react with a thiol group on the reaction partner,for example moiety V².

In another embodiment, the moiety RM is selected from

which makes it able to react with an amino group, e.g., a primary orsecondary amino group, on the reaction partner, for example moiety V².

In another embodiment, the moiety RM is selected from

which makes it able to react with an aldehyde group on the reactionpartner, for example moiety V².

The linking group L² in a compound of formula (III) represents theremainder of RM when the reactive moiety RM has reacted with V². Thisgroup then links the moiety V² with L. The group that remains may be abond, meaning that L² is absent. Typically, however, L² is a linkinggroup. When a compound of formula (III) is formed other than via acompound of formula (IV), L² does not represent the remainder of RM, butmay represent a similar or the same moiety and in addition be selectedfrom for example optionally substituted C₁₋₆ alkylene, C₁₋₆heteroalkylene, C₃₋₇ cycloalkylene, C₁₋₇ heterocycloalkylene, C₆₋₁₀arylene, and C₁₋₁₀ heteroarylene.

In one embodiment, the moiety L² is absent.

In another embodiment, the moiety L² is, without limitation,

In a further embodiment, the moiety L² is

The Moiety V²

The moiety V² is a functional moiety, which means that it addsadditional functionality to a compound of the invention.

In one embodiment, V² is a targeting moiety. In another embodiment, theV² moiety is a moiety that improves the pharmacokinetic properties of acompound of the invention. In yet another embodiment, the V² moiety is amoiety that causes accumulation of a compound of the invention at atarget site. In yet another embodiment, the V² moiety is a moiety thatimproves the aqueous solubility of a compound of the invention. In yetanother embodiment, the V² moiety is a moiety that increases thehydrophobicity of a compound of the invention. In yet anotherembodiment, the V² moiety is a moiety that reduces extravasation of acompound of the invention. In yet another embodiment, the V² moiety is amoiety that reduces excretion of a compound of the invention. In yetanother embodiment, the V² moiety is a moiety that reduces theimmunogenicity of a compound of the invention. In yet anotherembodiment, the V² moiety is a moiety that enhances the circulation timeof a compound of the invention. In yet another embodiment, the V² moietyis a moiety that enhances the ability of a compound of the invention tocross a biological barrier, e.g., a membrane, cell wall, or theblood-brain barrier. In yet another embodiment, the V² moiety is amoiety that enhances the ability of a compound of the invention tointernalize. In yet another embodiment, the V² moiety is a moiety thatcauses the compounds of the invention to aggregate. In yet anotherembodiment, the V² moiety is a moiety that reduces aggregation of acompound of the invention. In yet another embodiment, the V² moiety is amoiety that causes a compound of the invention to form micelles orliposomes. In yet another embodiment, the V² moiety is a moiety thatcauses complexation of a compound of the invention to another molecule,e.g., a biomolecule. In yet another embodiment, the V² moiety is apolynucleotide moiety that complexes with a complementary nucleotidesequence, for example RNA or DNA. In yet another embodiment, the V²moiety is a moiety that causes a compound of the invention to bind,associate, interact, or complex to another moiety, for example a(functionalized) surface or solid support.

In another embodiment, V² exhibits two or more different functions.

In one aspect of the invention, the moiety V² includes within its scopeany unit that binds or reactively associates or complexes with areceptor, a receptor complex, antigen, or other receptive moietyassociated with a given target cell population. V² can be any moleculethat binds to, complexes with, or reacts with a moiety of a cellpopulation sought to be therapeutically or otherwise biologicallymodified. The V² moiety acts to deliver the one or more moieties Z tothe particular target cell population with which V² reacts or to whichV² binds. Such V² moieties include, but are not limited to, aptamers,full-length antibodies and antibody fragments, lectins, biologicresponse modifiers, enzymes, vitamins, growth factors, steroids,nutrients, sugar residues, oligosaccharide residues, hormones, and anyderivatives thereof, or any combination of any of these. Upon binding,reactively associating, or complexing, the compounds of the inventionmay or may not be internalized. If internalization occurs,transformation and/or cleavage of V¹ preferably occur inside the targetcell.

Useful non-immunoreactive protein, polypeptide, or peptide V² moietiesinclude, but are not limited to, transferrin, epidermal growth factors(“EGF”), bombesin, gastrin and its derivatives, gastrin-releasingpeptide, platelet-derived growth factor, IL-2, IL-6, transforming growthfactors (“TGF”), such as TGF-a and TGF-P, tumor growth factors, vacciniagrowth factor (“VGF”), insulin and insulin-like growth factors I and II,lectins, and apoprotein from low density lipoprotein.

Useful polyclonal antibody V² moieties are heterogeneous populations ofantibody molecules. Various procedures well-known in the art may be usedfor the production of polyclonal antibodies to an antigen-of-interest.

Useful monoclonal antibody V² moieties are homogeneous populations ofantibodies to a particular antigen (e.g., a cancer cell antigen). Amonoclonal antibody (mAb) to an antigen-of-interest can be prepared byusing any technique known in the art which provides for the productionof monoclonal antibody molecules.

Useful monoclonal antibody V² moieties include, but are not limited to,human monoclonal antibodies, humanized monoclonal antibodies, orchimeric human-mouse (or other species) monoclonal antibodies. Humanmonoclonal antibodies may be made by any of numerous techniques known inthe art.

The V² moiety can also be a bispecific antibody. Methods for makingbispecific antibodies are known in the art.

The V² moiety can be a functionally active fragment, derivative, oranalog of an antibody that immunospecifically binds to an antigen on atarget cell, e.g., a cancer cell antigen. In this regard, “functionallyactive” means that the fragment, derivative, or analog is able to elicitanti-anti-idiotype antibodies that recognize the same antigen that theantibody from which the fragment, derivative, or analog is derived,recognizes.

Other useful V² moieties comprise fragments of antibodies including, butnot limited to, F(ab′)₂ fragments, which contain the variable region,the light chain constant region, and the CHI domain of the heavy chain,which can be produced by pepsin digestion of the antibody molecule, andFab fragments, which can be generated by reducing the disulfide bridgesof the F(ab′)₂ fragments. Other useful V² moieties are heavy chain andlight chain dimers of antibodies, or any minimal fragment thereof suchas Fvs or single chain antibodies (SCAs), domain antibodies, anticalins,affibodies, nanobodies, and any other molecules with the same, similar,or comparable specificity as the parent antibody.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are usefulV² moieties. A chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine monoclonal and a humanimmunoglobulin constant region. Humanized antibodies are antibodymolecules from non-human species having one or more complementaritydetermining regions (CDRs) from the non-human species and a frameworkregion from a human immunoglobulin molecule.

Completely human antibodies are particularly desirable as V² moieties.Such antibodies can for example be produced using transgenic mice thatare incapable of expressing endogenous immunoglobulin heavy and lightchains genes, but which can express human heavy and light chain genes.In other embodiments, the V² moiety is a fusion protein of an antibody,or a functionally active fragment or derivative thereof, for example onein which the antibody is fused via a covalent bond (e.g., a peptidebond) at either the N-terminus or the C-terminus to an amino acidsequence of another protein (or portion thereof, preferably at least a10, 20, or 50 amino acid portion of the protein) that is not theantibody. Preferably, the antibody or fragment thereof is covalentlylinked to the other protein at the N-terminus of the constant domain.

The V² moiety antibodies include analogs and derivatives that aremodified, i.e., by the covalent attachment of any type of molecule aslong as such covalent attachment permits the antibody to retain itsantigen binding immunospecificity. For example, but not by way oflimitation, derivatives and analogs of antibodies include those thathave been further modified, e.g., by glycosylation, acetylation,pegylation, disulfide reduction, phosphylation, amidation,derivatization by known protecting or blocking groups, proteolyticcleavage, linkage to an other protein, etc. Additionally, the analog orderivative can contain one or more unnatural amino acids.

The V² moiety antibodies include antibodies having modifications (e.g.,substitutions (for example cysteine to serine), deletions, or additions)in amino acid residues that interact with Fc receptors. In particular,they include antibodies having modifications in amino acid residuesidentified as involved in the interaction between the Fc domain and theFcRn receptor. Modifications may also be introduced to be able to couplethe antibody to linker-agent conjugates at specific positions on theantibody.

In a specific embodiment, an antibody immunospecific for a cancer ortumor antigen is used as a V² moiety in accordance with the compounds,compositions, and methods of the invention.

Antibodies immunospecific for a cancer cell antigen can be obtainedcommercially or produced by any method known to one of skill in the art,such as chemical synthesis or recombinant expression techniques. Thenucleotide sequences encoding antibodies immunospecific for a cancercell antigen can be obtained, e.g., from the GenBank database or adatabase like it, a commercial or other source, literature publications,or by routine cloning and sequencing.

Examples of antibodies available for the treatment of cancer include,but are not limited to, HERCEPTIN (trastuzumab; Genentech, CA) which isa humanized anti-HER2 monoclonal antibody for the treatment of patientswith metastatic breast cancer; RITUXAN (rituximab; Genentech, CA), whichis a chimeric anti-CD20 monoclonal antibody for the treatment ofpatients with non-Hodgkin's lymphoma; OvaRex (oregovomab; AltaRexCorporation, MA) which is a murine antibody for the treatment of ovariancancer; Panorex (edrecolomab; Glaxo Wellcome, NC) which is a murineIgG_(2a) antibody for the treatment of colorectal cancer; IMC-BEC2(mitumomab; ImClone Systems, NY) which is a murine IgG antibody for thetreatment of lung cancer; IMC-C225 (crbitux; Imclone Systems, NY) whichis a chimeric IgG antibody for the treatment of head and neck cancer;Vitaxin (MedImmune, MD) which is a humanized antibody for the treatmentof sarcoma; Campath I/H (alemtuzumab, Leukosite, MA) which is ahumanized IgG₁ antibody for the treatment of chronic lymphocyticleukemia (CLL); SGN-70 (Seattle Genetics, WA) which is a humanizedanti-CD70 antibody for the treatment of hematologic malignancies; SmartMI95 (Protein Design Labs, CA) which is a humanized IgG antibody for thetreatment of acute myeloid leukemia (AML); LymphoCide (epratuzumab,Immunomedics, NJ) which is a humanized IgG antibody for the treatment ofnon-Hodgkin's lymphoma; SGN-33 (Seattle Genetics, WA) which is ahumanized anti-CD33 antibody for the treatment of acute myeloidleukemia; Smart ID 10 (Protein Design Labs, CA) which is a humanizedantibody for the treatment of non-Hodgkin's lymphoma; Oncolym(Techniclone, CA) which is a murine antibody for the treatment ofnon-Hodgkin's lymphoma; Allomune (BioTransplant, CA) which is ahumanized anti-CD2 mAb for the treatment of Hodgkin's disease ornon-Hodgkin's lymphoma; anti-VEGF (Genentech, CA) which is a humanizedantibody for the treatment of lung and colorectal cancers; SGN-40(Seattle Genetics, WA) which is a humanized anti-CD40 antibody for thetreatment of multiple myeloma; SGN-30 (Seattle Genetics, WA) which is achimeric anti-CD30 antibody for the treatment of Hodgkin's disease;CEAcide (Immunomedics, NJ) which is a humanized anti-CEA antibody forthe treatment of colorectal cancer; IMC-1C11 (ImClone Systems, NJ) whichis an anti-KDR chimeric antibody for the treatment of colorectal cancer,lung cancers, and melanoma; and Cetuximab (ImClone Systems, NJ) which isan anti-EGFR chimeric antibody for the treatment of epidermal growthfactor positive cancers.

Other antibodies useful in the treatment of cancer include, but are notlimited to, antibodies against the following antigens: CA125, CA15-3,CA19-9, L6, Lewis Y, Lewis X, alpha fetoprotein, CA 242, placentalalkaline phosphatase, prostate specific antigen, prostatic acidphosphatase, epidermal growth factor receptors, interleukin receptors,CanAg, PEM, AFP, HER2, EGFR, VEGFR1, VEGFR2, MAGE-1, LUCA, LUCA2,MAGE-2, MAGE-3, MAGE-4, anti-transferrin receptor, Eph receptor tyrosinekinases, PSCA, CLL-1, EphA2, EphB2, FLT3, p97, MUC1-KLH, MUC18, MUC16,PSMA, EpCAM, CTLA4, CEA, GD2, gp100, GD3 ganglioside, GPC-3, MARTI, PSA,IL-2 receptor, CD2, CD4, CD20, CD44, CD30, CD55, CD19, CD79, CD52, CD25,CD46, CD56, CD7, CD74, CD133, CD80, CD140b, CD33, CD22, HLA-DR,HLA-DR10, human chorionic gonadotropin, CD38, CD40, CD70, mucin, P21,MPG, and Neu oncogene product. Many other internalizing ornon-internalizing antibodies that bind to tumor-associated antigens canbe used in this invention, some of which have been reviewed¹⁶.

In some embodiments, the antibody is an anti-nuclear antibody or anantibody that can bind to a receptor or receptor complex expressed on atarget cell. The receptor or receptor complex can comprise animmunoglobulin gene superfamily member, an integrin, a chemokinereceptor, a TNF receptor superfamily member, a cytokine receptor, amajor histocompatibility protein, a complement control protein, or alectin.

In another specific embodiment, an antibody immunospecific for anantigen associated with an autoimmune disease is used as a V² moiety inaccordance with the compounds, compositions, and methods of theinvention.

In another specific embodiment, an antibody immunospecific for a viralor microbial antigen is used as a V² moiety in accordance with thecompounds, compositions, and methods of the invention. As used herein,the term “viral antigen” includes, but is not limited to, any viralpeptide or polypeptide protein that is capable of eliciting an immuneresponse. As used herein, the term “microbial antigen” includes, but isnot limited to, any microbial peptide, polypeptide, protein, saccharide,polysaccharide, or lipid that is capable of eliciting an immuneresponse.

New antibodies are continually being discovered and developed, and thepresent invention provides that these new antibodies may also beincorporated into a compound of this invention.

V² can react with the reactive moiety RM via for example a heteroatom onV². Heteroatoms that may be present on V² include, without limitation,sulfur (in one embodiment, from a sulfhydryl group), oxygen (in oneembodiment, from a carboxyl or hydroxyl group), and nitrogen (in oneembodiment, from a primary or secondary amino group). V² may also reactvia for example a carbon atom (in one embodiment, from a carbonylgroup). These atoms can be present on V² in V²'s natural state, forexample a naturally occurring antibody, or can be introduced into V² via(chemical) modification.

Free sulfhydryl groups can be generated in an antibody or antibodyfragment by reduction of the antibody (fragment) with a reducing agentsuch as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphinc (TCEP). Inthis way, modified antibodies can be obtained that can have from 1 toabout 20 sulfhydryl groups, but typically between about 1 and about 9sulfhydryl groups.

Alternatively, V² can have one or more carbohydrate groups that can bechemically modified to have one or more sulfhydryl groups. As anotheralternative, sulfhydryl groups can be generated by reaction of aminogroups, for example from lysine moieties, on V² with 2-iminothiolane(Traut's reagent) or another sulfhydryl-generating reagent.

In one embodiment, the V² moiety is a receptor-binding moiety.

In another embodiment, the V² moiety is an antibody or an antibodyfragment.

In another embodiment, the V² moiety is a monoclonal antibody or afragment thereof.

In one embodiment, V² has one or more sulfhydryl groups and V² reactswith one or more RM moieties of compounds of formula (IV) via one ormore of these sulfhydryl groups' sulfur atoms to form a compound offormula (III).

In yet another embodiment, V² contains one or more disulfide bonds thatcan be chemically reduced to sulfhydryl groups (two for each disulfidebond), which can then be reacted with one or more reactive moieties RM.

In another embodiment, V² contains about 1 to about 3 sulfhydryl groups,which can be reacted with one or more reactive moieties RM.

In another embodiment, V² contains about 3 to about 5 sulfhydryl groups,which can be reacted with one or more reactive moieties RM.

In another embodiment, V² contains about 7 to about 9 sulfhydryl groups,which can be reacted with one or more reactive moieties RM.

In another embodiment, V² can have one or more carbohydrate groups thatcan be chemically modified to have one or more sulfhydryl groups. V²reacts with RM moieties via these one or more sulfhydryl groups' sulfuratoms.

In another embodiment, V² can have one or more lysine groups that can bechemically modified to have one or more sulfhydryl groups, which can bereacted with one or more reactive moieties RM.

Reactive moieties that can react with a sulfhydryl group include, butare not limited to, carbamoyl halide, acyl halide, α-haloacetamide,halomethyl ketone, vinyl sulfone, maleimide, and 2-disulfanylpyridine.

In yet another embodiment, V² can have one or more carbohydrate groupsthat can be oxidized to provide one or more aldehyde groups. Thecorresponding aldehyde(s) can then react with one or more reactivemoieties RM. Reactive moieties that can react with a carbonyl group onV² include, but are not limited to, hydrazine, hydrazide, amine, andhydroxylamine.

In yet another embodiment, V² can have one or more amino groups, e.g.,from lysine residues, which can be reacted with one or more reactivemoieties RM. Reactive moieties that can react with an amino groupinclude, but are not limited to, carbamoyl halide, α-haloacetamide, acylhalide, aldehyde, sulfonyl chloride, alkyl halide, alkyl sulfonate,isocyanate, and isothiocyanate.

A conjugate of formula (III) may exist as a mixture, wherein eachcomponent of the mixture has a different q value. For example, thecompound may exist as a mixture of two separate compounds, one compoundwherein q is 3 and another compound wherein q is 4. When analyzing thecompound of formula (III) it is understood that q may be the (rounded)average number of L²-L(-(V¹—Y))_(p)(Z)_(z/q) units per V² moiety.Furthermore, for a given q, the compound may exist as a mixture of(constitutional) isomers as the q L²-L(-(V¹—Y))_(p)(Z)_(z/q) moietiesmay be connected to distinct sets of functional groups on V². It shouldbe noted that the number of Z moieties in each unit only equals z/q ifall units are the same and/or contain the same number of Z moieties.

In one embodiment, the V² moiety is connected to L² via a sulfur atom ofV².

In another embodiment, the V² moiety is connected to L² via a sulfuratom and q ranges from about 1 to about 20.

In another embodiment, the V² moiety is connected to L² via a sulfuratom and q ranges from about 1 to about 9.

In another embodiment, the V² moiety is connected to L^(z) via a sulfuratom and q ranges from about 1 to about 3.

In another embodiment, the V² moiety is connected to L² via a sulfuratom and q is about 2.

In another embodiment, the V² moiety is connected to L² via a sulfuratom and q ranges from about 3 to about 5.

In another embodiment, the V² moiety is connected to L² via a sulfuratom and q is about 4.

In another embodiment, the V² moiety is connected to L² via a sulfuratom and q ranges from about 7 to about 9.

In another embodiment, the V² moiety is connected to L² via a sulfuratom and q is about 8.

In one embodiment, a compound of formula (III) exists as a mixture ofseparate compounds.

In one embodiment, a compound of formula (III) exists as a mixture ofseparate compounds wherein q for three compounds is 1, 2, and 3,respectively.

In one embodiment, a compound of formula (HI) exists as a mixture ofseparate compounds wherein q for three compounds is 3, 4, and 5,respectively.

In one embodiment, a compound of formula (III) exists as a mixture ofseparate compounds wherein q for three compounds is 5, 6, and 7,respectively.

In one embodiment, a compound of formula (III) exists as a mixture ofseparate compounds wherein q for three compounds is 7, 8, and 9,respectively.

In another embodiment, the V² moiety is connected to L² via a nitrogenatom of V².

In yet another embodiment, the V² moiety is connected to L² via a carbonatom of V².

In another aspect of this invention, the V² moiety includes any unitthat causes accumulation of compounds of the invention at the targetsite or in the vicinity thereof by a mechanism other than binding orreactively associating or complexing with a receptor, antigen, or otherreceptive moiety associated with a given target site, e.g., a targetcell population. One way to achieve this is for example to use a largemacromolecule as a V² moiety, which targets to solid tumor tissuethrough the enhanced permeability and retention (EPR) effect. Ringsdorfreported use of polymers to target antitumor agents to tumors.¹⁷ Throughthis EPR effect, macromolecules passively accumulate in solid tumors asa consequence of the disorganized pathology of angiogenic tumorvasculature with its discontinuous endothelium, leading tohyperpermeability to large macromolecules, and the lack of effectivetumor lymphatic drainage.

The V² moiety may for example be a branched or unbranched polymer, suchas for example poly[N-(2-hydroxypropyl)methacrylamide](HPMA),poly(2-hydroxyethyl methacrylate) (HEMA), polyglutamic acid orpoly-L-glutamic acid (PG), carboxymethyldextran (CMDex), a polyacetal,chitosan, a polypeptide, an oligoethylene glycol or polyethylene glycol(PEG), or a copolymer, such as an HPMA copolymer, an HPMA-methacrylicacid copolymer, a HEMA-methacrylic acid copolymer, a CMDex copolymer, aβ-cyclodextrin copolymer, a PEG copolymer, or a poly(lactic-co-glycolic)acid copolymer.¹⁸ In this document both polymer and copolymer arereferred to as polymer.

The polymer may be connected to L² via any suitable functional group,which can be located at one or both ends of the polymer, meaning that inthe conjugate q ranges from 1 to 2, or alternatively, the functionalgroups may (also) be located on groups pendant on the polymer such thatL² is (also) connected to the polymer via these pendant groups with qtypically ranging from 1 to about 1000. Optionally, the polymer may alsocontain an additional targeting group that can bind or reactivelyassociate or complex with a receptive moiety, e.g., an antibody orantibody derivative, bonded to the polymer either via a pendant group orend group, such that improved targeting to the target site is achieved.

Alternatively, the V² moiety may be a dendrimer or a protein or proteinfragment, e.g., albumin, which has no targeting properties except forits ability to accumulate at the target site because of its size ormolecular weight.

In one embodiment, the V² moiety is a polymer.

In another embodiment, the V² moiety is a polymer and q ranges from 1 toabout 1000.

In other embodiments, the V² moiety is a polymer and q ranges from 1 toabout 500 or 400 or 300 or 200 or 100 or less than 100.

In another embodiment, the V^(z) moiety is a polymer and q ranges from 1to 2.

In a specific embodiment, the V² moiety is an oligoethylene glycol or apolyethylene glycol or a derivative thereof.

In another embodiment, the V² moiety is a dendrimer, a protein, or aprotein fragment.

In another embodiment, V² is absent.

In another embodiment, the V² moiety is a moiety that is able totransport the conjugate across a biological barrier, e.g., a cellmembrane, either with or without prior binding, associating, orcomplexing with a receptor or receptor complex. In one embodiment, theV² moiety is a Tat peptide or a derivative, fragment, or analog thereof,or a moiety that has similar transmembrane delivery properties. Inanother embodiment, the V² moiety is protein or protein fragment, anantibody or an antibody fragment, a receptor-binding or peptide vectormoiety, or a polymeric or dendritic moiety, or any combination thereof,to which is attached a Tat peptide or a derivative, fragment, or analogthereof, or a moiety that has similar transmembrane delivery properties.

Thus, in one aspect of the invention, the moiety V² is a targetingmoiety and is selected from the group consisting of a protein or proteinfragment, an antibody or an antibody fragment, a receptor-binding orpeptide vector moiety, and a polymeric or dendritic moiety, and anycombination thereof.

In another aspect of the invention, the V² moiety is a moiety thatimproves the pharmacokinetic properties of a conjugate of the invention.For example, the moiety V² can be chosen such that the water solubilityof the conjugate is (further) improved. This can be achieved by choosingV² to be a hydrophilic moiety. Alternatively, the V² moiety can be usedto for example increase the residence time of the compound in thecirculation, to reduce extravasation and/or excretion, to reduceaggregation, and/or to reduce the immunogenicity of the compound. Thismay for example be achieved by choosing V² to be a polyethylene glycolor oligoethylene glycol or derivative thereof. When the moiety V² is amoiety that improves the pharmacokinetic properties of a compound of theinvention and V¹ is a moiety that can be cleaved or transformedaspecifically and there are no V^(1′) and V^(2′) moieties, the compoundsolely serves to improve the (pharmacokinetic) properties of the one ormore Z moieties.

In one embodiment, V² is a moiety that improves the pharmacokineticproperties and V¹ is a moiety that can be cleaved or transformedspecifically.

In another embodiment, V² is an oligoethylene glycol or a polyethyleneglycol or a derivative thereof and V¹ is a moiety that can be cleaved ortransformed specifically.

In another embodiment, V² is a moiety that improves the pharmacokineticproperties and V¹ is a moiety that can be cleaved or transformedaspecifically.

In another embodiment, V² is an oligoethylene glycol or a polyethyleneglycol or a derivative thereof and V¹ is a moiety that can be cleaved ortransformed aspecifically.

In another embodiment, V² is an oligoethylene glycol or a polyethyleneglycol or a derivative thereof and V¹ is a moiety that can be cleaved byubiquitous enzymes.

In another embodiment, V² is an oligoethylene glycol or a polyethyleneglycol or a derivative thereof and V¹ is a hydrolyzable moiety.

In one aspect of this invention, the V² moiety is represented by formula(VI):

wherein V^(2*), L^(2*), L*, V^(1*), Y*, p*, q*, and z* have the samemeaning as V², L², L, V¹, Y, p, q, and z, respectively, as defined inthis document, except that Y* is connected to L². It should be notedthat z* actually equals q. When a compound of formula (III) contains aV² moiety represented by formula (VI), the one or more L² moieties arethus connected to Y*. It should be understood that in this document,whenever V², L², L, V¹, Y, p, q, or z is mentioned, the same can applyfor each V^(2*), L^(2*), L*, V^(1*), Y*, p*, q*, or z*, respectively.

Use of a V² moiety of formula (VI) in a conjugate of formula (III)implicates that two conditionally-cleavable orconditionally-transformable moieties may be present in the samepromoiety, and therefore two separate cleavages/transformations may berequired to completely remove the promoiety. The requirement that twodifferent conditions need to have been met before one or more Z arereleased might favorably affect the properties of the conjugate. Forinstance, it may increase the targeting efficiency of the conjugate. Thetwo transformations/cleavages may occur at differentextracellular/intracellular locations. The moiety to be removed by thesecond cleavage or as a consequence of the second transformation may beused in this instance to help transport Z from a first extracellular orintracellular location to a second extracellular or intracellularlocation.

It will be apparent that a V² moiety of formula (VI) cannot only beuseful in conjugates of a compound of formula (I) or (II), but may beused in similar conjugates of other therapeutic agents, diagnosticmoieties, and the like.

It should be understood that the functional moiety V² can have severalfunctional properties combined. For example, V² can be a moiety thatimproves the pharmacokinetic properties of a compound of this inventionand at the same time be or contain a targeting moiety.

Conjugates of this invention may contain one or more promoieties. Thesepromoieties may be the same or different. The presence of two or morepromoieties may favorably affect the properties of the conjugate. Forinstance, it may improve the water solubility and/or increase thetargeting efficiency of the conjugate. In one embodiment, when there aretwo or more promoieties, said promoieties are different from each other.The two or more different promoieties may have different functions andmay be removed under different conditions and at differentextracellular/intracellular locations.

In one embodiment, there is one promoiety linked to Z.

In another embodiment, there is one promoiety linked to Z via X¹.

In another embodiment, there are two promoieties linked to Z.

In another embodiment, there are two promoieties linked to Z, of whichone is connected via X¹.

In yet another embodiment, there are three promoieties linked to Z.

In yet another embodiment, there are three promoieties linked to Z, ofwhich one is connected via X¹.

In one embodiment, a compound of formula (III) is represented by acompound of formula (III-1) or (III-2):

In another embodiment, a compound of formula (III) is represented by acompound of formula (III-3) or (III-4):

wherein Y′ is connected to an atom being part of R⁸, R⁹, R¹⁰, or R¹¹.

In one embodiment, p is an integer from 1 (included) to 128 (included).In another embodiment, q is an integer from 1 (included) to 1000(included). In other embodiments, p is an integer from 1 (included) to64 (included) or 32 (included) or 16 (included) or 8 (included) or 4(included) or 2 (included). In other embodiments, q is an integer from 1(included) to 500 (included) or 400 (included) or 300 (included) or 200(included) or 100 (included) or 16 (included) or 8 (included) or 6(included) or 4 (included) or 2 (included).

In one embodiment, if more than 1 promoiety is connected to a first Zand in one of the promoieties there is more than one attachment site forZ moieties, then the other ones of said promoieties connected to saidfirst Z each contain a single attachment site for a Z moiety.

In one embodiment, a compound of formula (III) is represented by

In one embodiment, p in a compound of formula (IIIa) is 1.

In another embodiment, in a compound of formula (IIIa) p is 1 and zequals q.

In another embodiment, a compound of formula (IIIa) is represented by

or by an isomer, or by a mixture of isomers, wherein R^(1a), R^(2b),R^(5b), R^(6b), R⁸, R⁹, and R¹⁰ are as previously defined, (AA)_(aa), isselected from valylcitrulline, valyllysine, phenylalanyllysine,alanylphenylalanyllysine, and D-alanylphenylalanyllysine, ss is 1 or 2,LL is selected from

qq ranges from 1 to 20, rr and rr′ each independently range from 1 to 4,and Ab is an antibody or a fragment or derivative thereof.

In another embodiment, a compound of formula (III) is represented by

In one embodiment, p* in a compound of formula (IIIa*) is 1.

In another embodiment, in a compound of formula (IIIa*) p* is 1 and z*equals q*.

In another embodiment, a compound of formula (III) is represented by

In one embodiment, p in a compound of formula (IIIb) is 1.

In another embodiment, a compound of formula (III) is represented by

In one embodiment, p* in a compound of formula (IIIb*) is 1.

In another embodiment, in a compound of formula (IIIb*) p* is 1 and z*equals q*.

In another embodiment, V¹ in a compound of formula (IIIb*) is anenzyme-cleavable substrate. In a further embodiment, V¹ can be cleavedby an intracellular enzyme. In another embodiment, V¹ is an optionallysubstituted N,N-dialkylaminocarbonyl group wherein the two alkyl groupsmay be the same or different and optionally be connected to each otherto form an optionally substituted heterocycle. In yet anotherembodiment, V¹ is piperazinocarbonyl.

In another embodiment, a compound of formula (IIIb*) is represented by

or by an isomer, or by a mixture of isomers, wherein R^(1a), R^(2b),R^(5b), R^(6b), R⁸, R⁹, and R¹⁰ are as previously defined, (AA)_(aa) isselected from valylcitrulline, valyllysine, phenylalanyllysinealanylphenylalanyllysine, and D-alanylphenylalanyllysine, ss is 1 or 2,LL is selected from

qq ranges from 1 to 20, rr and rr′ each independently range from 1 to 4,and Ab is an antibody or a fragment or derivative thereof.

In another embodiment, a compound of formula (III) is represented by

In yet another embodiment, a compound of formula (III) is represented byV¹—Z  (IIId).

In one embodiment, a compound of formula (IIId) is represented by

or by an isomer, or by a mixture of isomers, wherein R^(1a), R^(2b),R^(5b), R^(6b), R⁸, R⁹, and R¹⁰ are as previously defined, ss is 0, 1,or 2, (AA)_(aa) is selected from valylcitrulline, valyllysine,phenylalanyllysine, alanylphenylalanyllysine, andD-alanylphenylalanyllysine, the p-aminobcnzyloxycarbonyl group—or the(AA)_(aa) group if ss is 0—is connected to an oxygen, sulfur, ornitrogen atom in R⁸, R⁹ or R¹⁰, LL is selected from

qq′ ranges from 1 to 20, rr and rr′ each independently range from 1 to4, and Ab is an antibody or a fragment or derivative thereof.

In yet another embodiment, a compound of formula (III) is represented byV²-L²-L-Z  (IIIe).

In one embodiment, a compound of formula (IIIe) is represented by

or by an isomer, or by a mixture of isomers, wherein R^(1a), R^(2b),R^(5b), R^(6b), R⁸, R⁹, and R¹⁰ are as previously defined, (AA)_(aa) isselected from valylcitrulline, valyllysine, phenylalanyllysine,alanylphenylalanyllysine, and D-alanylphenylalanyllysine, LL is selectedfrom

qq ranges from 1 to 20, rr and rr′ each independently range from 1 to 4,R⁹⁰ is selected from H and C₁₋₃ alkyl, and Ab is an antibody or afragment or derivative thereof.

In another embodiment, a compound of formula (IIIe) is represented by

or by an isomer, or by a mixture of isomers, wherein R^(1a), R^(2b),R^(5b), R^(6b), R⁸, R⁹, and R¹⁰ are as previously defined, (AA), isselected from valylcitrulline, valyllysine, phenylalanyllysine,alanylphenylalanyllysine, and D-alanylphenylalanyllysine, LL isconnected to an oxygen, sulfur, or nitrogen atom in R⁸, R⁹ or R¹⁰, LL isselected from

qq′ ranges from 1 to 20, rr and rr′ each independently range from 1 to4, R⁹⁰ is selected from H and C₁₋₃ alkyl, and Ab is an antibody or afragment or derivative thereof.

In one embodiment, a compound of formula (III) is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of formula (III) is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of formula (III) is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of formula (III) is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of formula (III) is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of formula (III) is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of formula (III) is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of formula (III) is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of this invention is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of this invention is represented by

or by an isomer, or by a mixture of isomers.

In another embodiment, a compound of this invention is represented by

or by an isomer, or by a mixture of isomers.

Synthesis of Compounds of the Invention

As described in somewhat more detail below, compounds of formulae(I)-(IV) can be conveniently prepared in a way for some part analogousto compounds reported in WO 01/83448, WO 2004/043493, and WO 02/083180.

In one embodiment, a compound of formula (I) or (II) is used to preparea compound of formula (III). In another embodiment, a compound offormula (I) or (II) is used to prepare a compound of formula (IV). Inanother embodiment, a compound of formula (IV) is used to prepare acompound of formula (III). In another embodiment, a compound of formula(III) wherein V¹ is a protecting group is used to prepare anothercompound of formula (III) wherein V¹ is an in vivo removable moiety.

Several agents have been prepared in good yields. For example, agent 4was prepared in 6 steps from naphthalene 1, as depicted in FIG. 2.Alkylation of 1 followed by a radical cyclization step provided racemic2. The enantiomers were then separated on a Chiralpak IA HPLC column. Asthere is only one chiral center in compound 2, the theoretical yieldwith which each enantiomer can be obtained after the radical cyclizationstep amounts to 50%, which is twice as much as the maximum yield forsimilar enantiopure compounds containing two different substituents onthe carbon bearing the leaving group, as for example compound 5 in FIG.3.

Removal of the benzyl protecting group in 2 with palladium and ammoniumformate as the hydrogen donor, subsequent removal of the tert-butylprotecting group, and final coupling to indole 3 afforded agent 4 ingood yield.

Agent 7 was prepared from 5 according to a similar process using indole6 (FIG. 3). Compound 9 was prepared from 5 using a slightly differentapproach (FIG. 4). The benzyl protecting group was only removed aftercoupling of indole 8 to deprotected 5.

Agents 11 (FIG. 5) and 12 (FIG. 6) were synthesized from 10 and indoles6 and 8, respectively, in a manner largely analogous to the preparationof 7 from 5.

Agents such as 4, 7, 9, 11, and 12 can be incorporated into linker-drugconjugates and conjugates following established procedures. Someexemplary structures of linker-agent conjugates have been depicted inFIG. 7.

Uses, Methods, and Compositions

In one aspect, this invention relates to use of a compound of formula(I) or (II) for the preparation of a compound of formula (III).

In another aspect, this invention relates to use of a compound offormula (IV) for the preparation of a compound of formula (III).

In yet another aspect, this invention relates to use of a compound offormula (I) or (II) for the preparation of a compound of formula (IV).

In yet another aspect, this invention relates to use of a compound offormula (III) wherein V¹ is a protecting group for the preparation ofanother compound of formula (III) wherein V¹ is an in vivo removablemoiety.

In yet another aspect, the invention relates to the use of any of thecompounds defined above for the manufacture of a pharmaceuticalcomposition for the treatment of a mammal being in need thereof.

In one embodiment, the invention relates to the use of any of thecompounds defined above for the manufacture of a pharmaceuticalcomposition for the treatment of a tumor in a mammal.

The invention also relates to any of the compounds defined above as amedicament or an active component or active substance in a medicament.

In a further aspect, the invention relates to a process for preparing apharmaceutical composition containing a compound as defined above, toprovide a solid or a liquid formulation for administration orally,topically, or by injection. Such a method or process at least comprisesthe step of mixing the compound with a pharmaceutically acceptablecarrier.

In one embodiment, a compound of the invention is used to treat anillness characterized by undesired proliferation. In another embodiment,a compound of the invention is used to treat an illness characterized byundesired cell proliferation. In another embodiment, a compound of theinvention is used to treat a tumor. In yet another embodiment, acompound of the invention is used to treat an inflammatory disease. Inyet another embodiment, a compound of the invention is used to treat anautoimmune disease. In yet another embodiment, a compound of theinvention is used to treat a bacterial or microbial infection.

In a further embodiment, this invention relates to a method of treatinga mammal having an illness characterized by undesired (cell)proliferation with a compound of this invention. In another embodiment,this invention relates to a method of treating a mammal carrying a tumorwith a compound of this invention. In yet another embodiment, thisinvention relates to a method of treating a mammal having aninflammatory disease with a compound of this invention. In yet anotherembodiment, this invention relates to a method of treating a mammalhaving an autoimmune disease with a compound of this invention. In yetanother embodiment, this invention relates to a method of treating amammal having a bacterial or microbial infection with a compound of thisinvention.

In a further embodiment, the invention relates to a method of treating amammal being in need thereof, whereby the method comprises theadministration of a pharmaceutical composition comprising a compound ofthis invention to the mammal in a therapeutically effective dose.

In one embodiment, the invention relates to a method of treating orpreventing a tumor in a mammal, whereby the method comprises theadministration of a pharmaceutical composition comprising a compound ofthis invention to the mammal in a therapeutically effective dose.

In another embodiment, the invention relates to a method of treating orpreventing an inflammatory disease in a mammal, whereby the methodcomprises the administration of a pharmaceutical composition comprisinga compound of this invention to the mammal in a therapeuticallyeffective dose.

In another embodiment, the invention relates to a method of treating orpreventing an autoimmune disease in a mammal, whereby the methodcomprises the administration of a pharmaceutical composition comprisinga compound of this invention to the mammal in a therapeuticallyeffective dose.

In another embodiment, the invention relates to a method of treating orpreventing a bacterial or microbial infection in a mammal, whereby themethod comprises the administration of a pharmaceutical compositioncomprising a compound of this invention to the mammal in atherapeutically effective dose.

The invention also relates to pharmaceutical compositions comprising thecompounds of the invention as defined above. A compound of the inventionmay be administered in purified form together with a pharmaceuticalcarrier as a pharmaceutical composition. The preferred form depends onthe intended mode of administration and therapeutic application. Thepharmaceutical carrier can be any compatible, nontoxic substancesuitable to deliver the compounds of the invention to the patient.Pharmaceutically acceptable carriers are well known in the art andinclude, for example, aqueous solutions such as (sterile) water orphysiologically buffered saline or other solvents or vehicles such asglycols, glycerol, oils such as olive oil or injectable organic esters,alcohol, fats, waxes, and inert solids. A pharmaceutically acceptablecarrier may further contain physiologically acceptable compounds thatact for example to stabilize or to increase the absorption of thecompounds of the invention. Such physiologically acceptable compoundsinclude, for example, carbohydrates, such as glucose, sucrose, ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins, or other stabilizers orexcipients. One skilled in the art would know that the choice of apharmaceutically acceptable carrier, including a physiologicallyacceptable compound, depends, for example, on the route ofadministration of the composition. Pharmaceutically acceptableadjuvants, buffering agents, dispersing agents, and the like, may alsobe incorporated into the pharmaceutical compositions.

For oral administration, the active ingredient can be administered insolid dosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions.

Active component(s) can be encapsulated in gelatin capsules togetherwith inactive ingredients and powdered carriers, such as glucose,lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives,magnesium stearate, stearic acid, sodium saccharin, talcum, magnesiumcarbonate, and the like. Examples of additional inactive ingredientsthat may be added to provide desirable color, taste, stability,buffering capacity, dispersion, or other known desirable features arered iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide,edible white ink, and the like. Similar diluents can be used to makecompressed tablets. Both tablets and capsules can be manufactured assustained release products to provide for continuous release ofmedication over a period of hours.

Compressed tablets can be sugar-coated or film-coated to mask anyunpleasant taste and protect the tablet from the atmosphere, orenteric-coated for selective disintegration in the gastrointestinaltract. Liquid dosage forms for oral administration can contain coloringand flavoring to increase patient acceptance.

The compounds of the invention are however preferably administeredparenterally. Preparations of the compounds of the invention forparenteral administration must be sterile. Sterilization is readilyaccomplished by filtration through sterile filtration membranes,optionally prior to or following lyophilization and reconstitution. Theparenteral route for administration of compounds of the invention is inaccord with known methods, e.g. injection or infusion by intravenous,intraperitoneal, intramuscular, intraarterial, or intralesional routes.The compounds of the invention may be administered continuously byinfusion or by bolus injection. A typical composition for intravenousinfusion could be made up to contain 100 to 500 ml of sterile 0.9% NaClor 5% glucose optionally supplemented with a 20% albumin solution and 1mg to 10 g of the compound of the invention, depending on the particulartype of compound of the invention and its required dosing regime.Methods for preparing parenterally administrable compositions are wellknown in the art and described in more detail in various sources,including, for example, Remington's Pharmaceutical Science¹⁹.

A compound of the invention may also be used in combination therapy, inwhich a compound of this invention is used in combination with one ormore other therapeutic agents. Combination of two or more therapeuticsmay favorably affect treatment outcome. The agents may be administeredeither sequentially or concomitantly. Therefore, in one embodiment thisinvention relates to use of a compound of this invention or apharmaceutical composition comprising a compound of this invention incombination therapy.

The invention is further exemplified by the following examples. Theseexamples are for illustrative purposes only and are not intended tolimit the scope of the invention.

EXAMPLES Example 1 Preparation of Compound 2 from Compound 1

To a suspension of NaH (0.50 g, 12.5 mmol) in DMF (30 ml) was added asolution of compound 1 (2.38 g, 5.00 mmol) in DMF (20 ml) and themixture was stirred for 1 h. Then, 1,1,3-trichloroprop-1-ene (1.02 g,7.00 mmol) was added and the mixture was stirred for 2.5 h. A saturatedaqueous solution of NH₄Cl (100 mL) was slowly added and the mixture wasextracted twice with ethyl acetate. The combined organics were washedwith water and brine, dried over Na₂SO₄, filtered, and concentrated.Column chromatography (heptanes/EtOAc, 6:1 to 3:1) gave the alkylatedproduct (2.43 g, 4.15 mmol, 83%) as an orange foam. The alkylatedproduct (2.42 g, 4.14 mmol) was dissolved in toluene (60 ml) and thesolution was heated to 80° C. AIBN (0.170 g, 1.035 mmol) and TTMSS(1.132 g, 4.55 mmol) were then added and the mixture was stirred at 80°C. for 5 h. Then, the mixture was cooled to RT, water was added, thelayers were separated, and the aqueous layer extracted with ethylacetate. The combined organic layers were dried over Na₂SO₄, filtered,and concentrated. Crystallization from heptane gave a mixture ofenantiomers (1.423 g, 3.11 mmol, 75%) as a slightly brown solid.Separation of the enantiomers was carried out using a Chiralpak IA HPLCcolumn to afford compound 2 with high enantiopurity. —¹H NMR (300 MHz,CDCl₃), δ (ppm): 1.61 (9H, s, t-butyl), 4.20 (1H, m, H-2), 4.35 (1H, m,H-2), 4.48 (1H, m, H-1), 5.25 (2H, s, Bn), 6.33 (1H, d, J=2.1 Hz, H-10),7.32-7.55 (8H, m, H-7, H-8, H-9, Bn), 7.88 (1H, br. s, H-4), 8.30 (1H,d, J=8.4 Hz, H-6).

Example 2 Preparation of Agent 4 from Compound 2

A solution of compound 2 (74 mg, 0.161 mmol) in THF (5 ml) was warmed to45° C., after which palladium (10% on carbon, 34.4 mg, 0.032 mmol) andammonium formate (25% aqueous solution, 406 mg, 1.61 mmol) were added.The mixture was stirred for 2.5 h, cooled to RT, and filtered overCelite. The filtrate was concentrated and purified by columnchromatography to yield the debenzylated intermediate (60 mg, 0.163mmol) as a white semi-solid. This intermediate (45 mg, 0.122 mmol) wasdissolved in 4 N HCl in ethyl acetate (6 ml) and the solution wasstirred for 2 h. Then, the mixture was concentrated and dried in vacuo.The crude product was dissolved in dry DMF (4 ml) and the solution wascooled to 0° C. Indole 3 (45.2 mg, 0.159 mmol) and EDC.HCl (70.3 mg,0.367 mmol) were added. The mixture was stirred at RT for 18 h and thenconcentrated. The crude product was purified by column chromatography(DCM/MeOH, 19:1, 0.05% conc. HCl) to yield 4 (65 mg, 0.122 mmol, 99%) asa pale yellow solid. —¹H NMR (300 MHz, CDCl₃/CD₃OD), δ (ppm): 3.02 (6H,br. s, N(CH₃)₂), 3.54-3.63 (2H, m, H-2″), 4.42-4.53 (3H, m, H-1, H-1″),4.76 (1H, t, J=7.5 Hz, H-2), 5.03 (1H, d, J=8.1 Hz, H-2), 6.36 (1H, br.s, H-10), 7.04-7.12 (2H, m, H-3′, H-6′), 7.24 (1H, br. s, H-4′),7.40-7.70 (2H, m, H-8, H-7′), 7.56 (1H, t, J=6.0 Hz, H-7), 7.66 (1H, d,J=6.3 Hz, H-9), 7.82 (1H, br. s, H-4), 8.30 (1H, d, J=6.0 Hz, H-6); MS(ESI) m/z=498 (M+H⁺).

Example 3 Preparation of Agent 7 from Compound 5

A solution of compound 5 (54 mg, 0.123 mmol) in THF (7 ml) was warmed to45° C., after which palladium (10% on carbon, 96 mg, 0.090 mmol) andammonium formate (25% aqueous solution, 310 mg, 1.23 mmol) were added.The mixture was stirred for 0.5 h, cooled to RT, and filtered overCelite. The filtrate was concentrated and purified by columnchromatography to yield the debenzylated intermediate (41 mg, 0.118mmol) as a white semisolid. This intermediate (45 mg, 0.122 mmol) wasdissolved in 4 N HCl in ethyl acetate (5 ml) and the resultant solutionwas stirred for 2 h. Then, the mixture was concentrated and dried invacuo. The crude product was dissolved in dry DMF (6 ml) and thesolution was cooled to 0° C. Indole 6 (35.0 mg, 0.172 mmol) and EDC.HCl(66.1 mg, 0.345 mmol) were added. The mixture was stirred at RT for 18 hand then concentrated. The crude product was purified by columnchromatography (DCM/MeOH, 19:1, 0.05% conc. HCl) to yield 7 (23 mg,0.053 mmol, 46%) as a pale yellow solid. —¹H NMR (300 MHz, CDCl₃/CD₃OD),δ (ppm): 1.61 (3H, d, J=8.0 Hz, 10-CH₃), 2.66 (3H, s, Ac), 3.96 (1H, m,H-1), 4.51-4.63 (2H, m, H-2, H-10), 4.77 (1H, d, J=12.0, H-2), 7.20 (1H,s, H-3′), 7.36 (1H, t, J=8.0 Hz, H-7), 7.46-7.52 (2H, m, H-8, H-9), 7.66(1H, d, J=8.0 Hz, H-7′), 7.75 (1H, br. s, H-4), 7.93 (1H, dd, H-6′),8.24 (1H, d, J=8.0 Hz, H-6), 8.40 (1H, s, H-4′); MS (ESI) m/z=433(M+H⁺).

Example 4 Preparation of Agent 9 from Compound 5

Compound 5 (80 mg, 0.183 mmol) was dissolved in 4 N HCl in EtOAc (8 ml).The solution was stirred for 1 h and then concentrated. The crudeproduct was dried and then dissolved in dry DMF (4 ml). The solution wascooled to 0 OC and 8 (45.9 mg, 0.183 mmol) and EDC.HCl (105 mg, 0.548mmol) were added. The mixture was stirred at RT for 18 h and thenconcentrated. The crude product was purified by column chromatography(DCM/EtOAc, 19:1) to yield the benzyl-protected agent (65 mg, 0.114mmol, 62%) as a white solid. A solution of this intermediate (65 mg,0.114 mmol) in THF (5 ml) was warmed to 45° C., palladium (10% oncarbon, 24 mg, 0.226 mmol) and ammonium formate (25% aqueous solution,288 mg, 1.14 mmol) were added, and the resultant mixture was stirred for2 h. The mixture was cooled to RT and filtered over Celite. The filtratewas concentrated and the crude product purified by column chromatography(DCM/MeOH, 19:1, 0.1% HCl) to yield 9 (44 mg, 0.091 mmol, 80%) as a paleyellow solid. —¹H NMR (300 MHz, CDCl₃), δ (ppm): 1.60 (3H, d, J=6.6 Hz,10-CH₃), 3.85-3.92 (4H, m, H-1, OMe), 3.95 (3H, s, OMe), 4.11 (3H, s,OMe), 4.45-4.56 (2H, m, H-10, H-2), 4.78 (1H, dd, J₁=10.5 Hz, J₂=1.5 Hz,H-2), 6.87 (1H, s, H-4′), 7.01 (1H, s, H-3′), 7.38 (1H, m, H-8), 7.48(1H, m, H-7), 7.63 (1H, d, J=8.4, H-9), 7.98 (1H, br. s, H-4), 8.31 (1H,d, J=8.7 Hz, H-6); MS (ESI) m/z=481 (M+H⁺).

Example 5 Preparation of Agent 11 from Compound 10

A solution of compound 10 (67 mg, 0.153 mmol) in THF (10 ml) was warmedto 45° C., after which palladium (10% on carbon, 32.6 mg, 0.031 mmol)and ammonium formate (25% aqueous solution, 386 mg, 1.53 mmol) wereadded. The mixture was stirred for 2.5 h, cooled to RT, and filteredover Celite. The filtrate was concentrated, the crude product dissolvedin 4 N HCl in ethyl acetate (6 ml), and the resultant mixture stirredfor 2 h. Then, the mixture was concentrated and dried in vacuo to affordthe intermediate (26.2 mg, 0.106 mmol) as an off-white solid. Theintermediate (13.1 mg, 0.053 mmol) was dissolved in dry DMF (2 ml) andthe solution was cooled to 0° C. Indole 6 (11.8 mg, 0.058 mmol) andEDC.HCl (30.4 mg, 0.159 mmol) were added. The mixture was stirred at RTfor 18 h and then concentrated. The crude product was purified by columnchromatography (DCM/MeOH, 19:1, 0.05% conc. HCl) to yield 11 (13.3 mg,0.031 mmol, 58%) as a pale yellow solid. —¹H NMR (300 MHz, CDCl₃), δ(ppm): 2.61 (3H, s, Ac), 2.64 (3H, s, 9-Me), 3.07 (1H, t, J=10.8 Hz,H-10), 3.49 (1H, d, J=10.7 Hz, H-10), 4.06 (1H, m, H-1), 4.35 (1H, t,J=8.9 Hz, H-2), 4.60 (1H, d, J=10.2 Hz, H-2), 7.05 (1H, s, H-3′),7.30-7.38 (3H, m, H-7, H-8, H-4′), 7.85 (1H, m, H-7′), 8.18 (1H, m,H-6′), 8.29 (2H, d, J=7.2 Hz, H-4, H-6); MS (ESI) m/z=433 (M+H⁺).

Example 6 Preparation of Agent 12 from Compound 10

A solution of compound 10 (67 mg, 0.153 mmol) in THF (10 ml) was warmedto 45° C., after which palladium (10% on carbon, 32.6 mg, 0.031 mmol)and ammonium formate (25% aqueous solution, 386 mg, 1.53 mmol) wereadded. The mixture was stirred for 2.5 h, cooled to RT, and filteredover Celite. The filtrate was concentrated, the crude product dissolvedin 4 N HCl in ethyl acetate (6 ml), and the solution stirred for 2 h.Then, the mixture was concentrated and dried in vacuo to afford theintermediate (26.2 mg, 0.106 mmol) as an off-white solid. Theintermediate (13.1 mg, 0.053 mmol) was dissolved in dry DMF (2 ml) andthe solution was cooled to 0° C. Indole 8 (14.6 mg, 0.058 mmol) andEDC.HCl (30.4 mg, 0.159 mmol) were added. The mixture was stirred for 18h at RT and then concentrated. The crude product was purified by columnchromatography (DCM/MeOH, 19:1, 0.05% conc. HCl) to yield 12 (12.2 mg,0.025 mmol, 48%) as a pale yellow solid. —¹H NMR (300 MHz, CDCl₃), δ(ppm): 2.67 (3H, s, 9-Me), 3.13 (1H, t, J=11.4 Hz, H-10), 3.50 (1H, d,J=11.1 Hz, H-10), 3.91 (3H, s, OMe), 3.97 (3H, s, OMe), 4.16 (4H, m,H-1, OMe), 4.53 (1H, t, J=8 Hz, H-2), 4.66 (1H, d, J=10.2 Hz, H-2), 6.77(1H, s, H-4′), 6.88 (1H, d, J=2.1 Hz, H-3′), 7.24-7.33 (2H, m, H-7,H-8), 8.27 (1H, m, H-6), 8.52 (1H, s, H-4), 9.62 (1H, s, OH), 10.08 (1H,s, NH); MS (ESI) m/z=481 (M+H⁺).

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The invention claimed is:
 1. A compound, which is

wherein R^(1a) is selected from Cl and Br; R^(2b) is hydrogen; R^(5b) isselected from methyl, ethyl, propyl, isopropyl, nitro, F, Cl, Br,methoxy, amino, methylamino, hydroxymethyl, and dimethylamino; R^(6b) isselected from H, tert-butyl, and isopropyl; R⁹ and R¹⁰ are both hydrogenand R⁸ is selected from the group consisting of N(R^(h))C(O)R^(i),N(R^(h))C(O)OR^(i), and N(R^(h))C(O)N(R^(i))R^(j), wherein R^(h) is H,R^(i)and R^(j) are independently selected from H and optionallysubstituted C₃₋₁₅ cycloalkyl, C₁₋₁₅ heterocycloalkyl, C₆₋₁₅ aryl, orC₁₋₁₅ heteroaryl, one or more of the optional substitutents in R^(i)and/or R^(j) optionally being a water-soluble group, (AA)_(aa) isselected from valylcitrulline, valyllysine, phenylalanyllysine,alanylphenylalanyllysine, and D-alanylphenylalanyllysine, ss is 1 or 2,LL is selected from

qq ranges from 1 to 20, rr and rr′ each independently range from 1 to 4,and Ab is an antibody.
 2. A pharmaceutical composition comprising acompound according to claim 1 and a pharmaceutically acceptable carrier.3. The compound of claim 1, wherein said Ab is a full-length antibody,an F(ab′)₂ fragment thereof, or an Fab fragment thereof.
 4. The compoundof claim 3, wherein said Ab is a monoclonal antibody.
 5. The compound ofclaim 1, wherein R^(6b) is hydrogen.